Method of and improved composition for treating triterpene-responsive conditions, diseases or disorders

ABSTRACT

A method of treating a triterpene-responsive condition, disease or disorder in a subject by administration of an improved triterpene-based composition is provided. An improved triterpene-based composition comprising at least two or at least three triterpenes present at a molar ratio as described herein is also provided.

INCORPORATION BY REFERENCE

In compliance with 37 CFR 1.821-1.825, the instant application containsSequence Listings which have been submitted in electronic format via EFSand which are hereby incorporated by reference. The sequence informationcontained in electronic file named PBI14DIV_SEQ_ST25.txt, size 2 KB,created on Sep. 30, 2020, using Patent-in 3.5.1, and Checker 4.4.6 ishereby incorporated by reference in its entirety.

CROSS-REFERENCE TO EARLIER FILED APPLICATIONS

The present invention claims the benefit of and is a divisional of Ser.No. 16/184,628 filed Nov. 8, 2018, which is a continuation ofPCT/US2018/059818 filed Nov. 8, 2018, which is a continuation-in-part ofPCT/US2018/049358 filed Sep. 4, 2018, which claims the benefit of62/558,631 filed Sep. 14, 2017, and said Ser. No. 16/184,628 is acontinuation-in-part of PCT/US2018/049358, the entire disclosures of allof which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention concerns a method of treatingtriterpene-responsive conditions, disease or disorders with an improvedcomposition comprising a combination of triterpenes present at a molarratio as described herein. In particular, the invention concerns amethod of treatment by administration of a triterpene-based compositionto a subject in need thereof. The invention also includes pharmaceuticalcompositions containing the improved composition.

BACKGROUND OF THE INVENTION

Neurological diseases and disorders affect brain function. Many effortshave been made to develop curative or ameliorative therapies for thesediseases and disorders; however, no comprehensive or universallycurative therapy has been developed, even though there are numerouspharmacotherapeutic approaches that have been proven to be effectiveagainst various different diseases and disorders.

Huntington's disease (HD) is an inherited disease of the brain thataffects the nervous system. It is caused by a defective gene that ispassed from parent to child. The HD gene interferes with the manufactureof a particular protein known as ‘Huntington’ which appears to becrucial for proper brain development. The classic signs of HD includeemotional, cognitive and motor disturbances. Huntington's ischaracterized by jerky involuntary movements (chorea), but sometimescauses rigidity without abnormal movements, changes in using the limbs(apraxia), loss of control of bodily functions and dementia, including aprogressive deterioration of memory, speed of thought, judgment, andlack of awareness of problems and planning. There is no known cure forHuntington's disease. Although there are a number of medications to helpcontrol symptoms associated with HD such as emotional and movementproblems, there is no treatment to stop or reverse the course of thedisease. Huntington's disease has been recognized as a disease with ageneral membrane abnormality. A significantly elevated level andactivity (10 fold increase) of Na,K-ATPase has been observed inmembranes of erythrocytes and basal ganglia of Huntington's patientscompared to that of normal (Butterfield D A, Oeswein J Q, Prunty M E,Hisle K C, Markesbery W R). Increased sodium, potassium adenosinetriphosphatase activity in erythrocyte membranes in Huntington'sdisease. Ann Neurology, 4:60-62, 1978) fibroblast membranes obtainedfrom the skin of Huntington's disease patients (Schroeder F, Goetz I E,Roberts E, Membrane anomalies in Huntington's disease fibroblasts. J.Neurochem. 43: 526-539, 1984).

Alzheimer's disease is a form of dementia a neurodegenerative diseasethat damages the brain's intellectual functions (memory, orientation,calculation, etc.), but usually preserves its motor functions. InAlzheimer's disease, the mind gradually deteriorates, causing memoryloss, confusion, disorientation, impaired judgment and other problemsthat may affect a person's ability to perform normal daily activities.The type, severity, sequence and progression of mental changes varygreatly. There is no known cure for Alzheimer's disease and no known wayto slow its progression. For some people in the early or middle stagesof the disease, medication such as tacrine may alleviate some cognitivesymptoms. Aricept (donepezil) and Exelon (rivastigmine) are reversibleacetylcholinesterase inhibitors that are indicated for the treatment ofmild to moderate dementia of the Alzheimer's type. These drugs (calledcholinesterase inhibitors) work by increasing the brain's levels of theneurotransmitter acetylcholine, helping to restore communication betweenbrain cells. Some medications may help control behavioral symptoms suchas sleeplessness, agitation, wandering, anxiety, and depression. Thesetreatments are aimed at making the patient more comfortable. Although nomedication is known to cure Alzheimer's disease, cholinesteraseinhibitors may improve performance of daily activities, or lessenbehavioral problems. Medications for the treatment of Alzheimer'sdisease currently being tested include estrogens, nonsteroidalanti-inflammatory agents, vitamin E, selegiline (Carbex, Eldepryl) andthe botanical product Gingko biloba.

Triterpenes are known to possess a wide variety of therapeuticactivities. Some of the known triterpenes include oleanolic acid,ursolic acid, betulinic acid, bardoxolone, maslinic acid, and others.The therapeutic activity of the triterpenes has primarily been evaluatedindividually rather than as combinations of triterpenes.

Rong et al. (Pharm. Biol. (January 2011), 49(1), 78-85) suggestoleanolic acid might be suitable for attenuating ischemic stroke. So etal. (Arch. Pharm. Res. (June 2009), 32(6), 923-932) suggest oleanolicacid might be suitable for the prevention and treatment ofneurodegeneration in stroke. Li et al. (Brain Res. (February 2013),1497, 32-39) suggest ursolic acid might provide neuroprotection aftercerebral ischemia in mice. Garcia-Morales et al. (Arch. Pharm. Res.(July 2015), 38(7), 1369-1379) suggest that an extract of Bouvardiaternfolia should be further studied for treating Alzheimer's disease.Zhang et al. (Neuroscience Letters (2014), 579, 12-17) report thatursolic acid reduces oxidative stress following experimentalsubarachnoid hemorrhage. Qian et al. (Eur. J. Pharmacol. (2011), 670(1),148-153) report that maslinic acid protects cortical neurons againstoxygen-glucose deprivation-induced injury in rats. EP 2260851 A1 toConsejo Superior de Investigaciones Cientificas (Madrid, ES) suggeststhe use of oleanolic acid for the treatment of multiple sclerosis. Yooet al. (Molecules, (May 2012), 17(3), 3524-38) suggest the use ofterpenoids as anti-Alzheimer's disease therapeutics. Heo et al. (Mol.Cells (February 2002), 13(1), 5-11) suggest ursolic acid reduces amyloidbeta protein-induced oxidative cell death. Chung et al. (Mol. Cells(April 2001), 11(2), 137-143) suggest ursolic acid appears to be apotent inhibitor of acetylcholinesterase in Alzheimer's disease. US2007/0249711 A1 (Pub. Date. Oct. 25, 2007) to Choi et al. suggests theuse of oleanolic acid and ursolic acid for improving brain functions toprevent and treat mild cognitive impairment and dementia.

Oleanolic acid is in a class of triterpenoids typified by compounds suchas bardoxolone which have been shown to be potent activators of theinnate cellular phase 2 detoxifying pathway, in which activation of thetranscription factor Nrf2 leads to transcriptional increases in programsof downstream antioxidant genes containing the antioxidanttranscriptional response element (ARE). Bardoxolone itself has beenextensively investigated in clinical trials in inflammatory conditions;however, a Phase 3 clinical trial in chronic kidney disease wasterminated due to adverse events that may have been related to knowncellular toxicities of certain triterpenoids including bardoxolone atelevated concentrations.

Compositions containing triterpenes in combination with othertherapeutic components are found as plant extracts. Fumiko et al. (Biol.Pharm. Bull (2002), 25(11), 1485-1487) discloses the evaluation of amethanolic extract of Rosmarimus officinalis L. for treatingtrypanosomiasis. Addington et al. (U.S. Pat. Nos. 8,481,086, 9,220,778,9,358,293, US 20160243143 A1) disclose a supercritical fluid extract(SCF; PBI-05204) of Nerium oleander containing oleandrin and triterpenesfor the treatment of neurological conditions. Addington et al. (U.S.Pat. No. 9,011,937, US 20150283191 A1) disclose a triterpene-containingfraction (PBI-04711) of the SCF extract of Nerium oleander containingoleandrin and triterpenes for the treatment of neurological conditions.Jager et al. (Molecules (2009), 14, 2016-2031) disclose various plantextracts containing mixtures of oleanolic acid, ursolic acid, betulinicacid and other components. Mishra et al. (PLoS One 2016 25;11(7):e0159430. Epub 2016 Jul. 25) disclose an extract of Betula utilisbark containing a mixture of oleanolic acid, ursolic acid, betulinicacid and other components. Wang et al. (Molecules (2016), 21, 139)disclose an extract of Alstonia scholaris containing a mixture ofoleanolic acid, ursolic acid, betulinic acid and other components. L. eSilva et al. (Molecules (2012), 17, 12197) disclose an extract of Eriopeblanchetti containing a mixture of oleanolic acid, ursolic acid,betulinic acid and other components. Rui et al. (Int. J. Mol. Sci.(2012), 13, 7648-7662) disclose an extract of Eucalyptus globuluscontaining a mixture of oleanolic acid, ursolic acid, betulinic acid andother components. Ayatollahi et al. (Iran. J. Pharm. Res. (2011), 10(2),287-294) disclose an extract of Euphorbia microsciadia containing amixture of oleanolic acid, ursolic acid, betulinic acid and othercomponents. Wu et al. (Molecules (2011), 16, 1-15) disclose an extractof Ligustrum species containing a mixture of oleanolic acid, ursolicacid, betulinic acid and other components. Lee et al. (Biol. Pharm. Bull(2010), 33(2), 330) disclose an extract of Forsythia viridissimacontaining a mixture of oleanolic acid, ursolic acid, betulinic acid andother components. Wozniak et al. (Molecules (2015), 20, 20614-20641)disclose various therapeutic activities of ursolic acid. Liby et al.(Pharmacol. Rev. (2012), 64:972-1003) disclose various therapeuticactivities of synthetic oleanane triterpenoids.

Oleanolic acid (O or OA), ursolic acid (U or UA) and betulinic acid (Bor BA) are the three major triterpene components found in PBI-05204(PBI-23; a supercritical fluid extract of Nerium oleander) and PBI-04711(a triterpene-containing fraction 0-4 of PBI-05204). We (two of theinstant inventors) previously reported (Van Kanegan et al., in NatureScientific Reports (May 2016), 6:25626. doi: 10.1038/srep25626) on thecontribution of the triterpenes toward efficacy by comparing theirneuroprotective activity in a brain slice oxygen glucose deprivation(OGD) model assay at similar concentrations. We found that PBI-05204(PBI) and PBI-04711 (Fraction 0-4) provide neuroprotective activity(FIG. 1). We then evaluated the neuroprotective activity of the threemajor individual triterpenes and of uvaol (Uva) individually in the OGDassay on an equimolar basis (FIG. 5). We found that OA provides higheractivity than UA; whereas BA and Uva (uvaol) provide little to noactivity at the concentrations tested. We found that the activity of UAin this assay exhibited variable activity in a concentration dependentmanner. We postulated activation of nuclear factor erythroid 2 relatedfactor (Nrf2)-dependent antioxidant genes as a potential mechanism forthe underlying neuroprotective activity of PBI-04711 and the individualtriterpenes. Therefore, employing an ARE-luciferase promoter-reporterassay, we determined the ability of those compositions to activate theNrf2-ARE (antioxidant transcriptional response element) gene pathway inneurons using a corticostriatal primary neuronal co-culture systemcomposed of the neuronal and glial cell types as in the brain slice OGDassay. We found (FIGS. 2A-2D) that PBI-04711 increased expression ofcanonical target ARE genes (glutamate-cysteine ligase, catalytic subunit(Gclc); NAD(P)H:quinone oxidoreductase 1 (Nqo1); sulfiredoxinantioxidant protein (Srx); and heme oxygenase 1 (Hmox1)) via activationof the transcription factor NRF2 that mediates the cellular antioxidantdefense pathway. However, when comparing this activity of the individualtriterpenes to that of PBI-04711 (FIG. 3), we found that UA appeared tobe considerably more potent, as single agent, in inducing ARE geneexpression compared to BA and OA, meaning the induction of Srx and Hmox1is due more so to the activity of UA than of OA or BA, but UA stillexhibits lower activity in the neuroprotection OGD assay. We found thatwhile UA and BA are most active at gene expression, they are also verytoxic at concentrations that are just 2-3-fold higher thanconcentrations required to induce gene expression. This means that UAand BA have narrow therapeutic windows. Our prior results suggested thatUA and BA would likely be too toxic to achieve doses that would realizethe full ARE-inducing activity in vivo. Our prior results also suggestedthat OA was relatively inactive on its own, so it would be unlikely thatthe combination of triterpenes (in PBI-05204 and PBI-04711) at theirmolar ratios could achieve substantially improved neuroprotectiveactivity at doses that are not toxic at a cellular level.

U.S. Pat. Nos. 8,481,086, 9,220,778, 9,358,293, and US 2016-0243143 A1disclose the use of PBI-05204 for the treatment of neurologicalconditions. U.S. Pat. No. 9,011,937 and US 2015-0283191 A1 disclose theuse of PBI-04711 for the treatment of neurological conditions.

Compositions containing plural triterpenes in certain molar ratios havebeen reported to be undesirable. Compositions comprising ursolic acidand oleanolic acid were evaluated to determine their impact uponplatelet aggregation. Kim et el. (“Enhancement of platelet aggregationby ursolic acid and oleanolic acid” in Biomol. Therap. (2014), 22(3),254-259) reported that the triterpenes potentiated platelet aggregationand “need to be used with caution, especially in the population with apredisposition to cardiovascular events”.

None of the art suggests a composition containing a combination of threedifferent triterpenes selected from oleanolic acid, ursolic acid andbetulinic acid, nor use of such a composition for the treatment oftriterpene-responsive conditions, diseases, or disorders, wherein thetriterpenes are present in the molar ratios as defined herein. None ofthe art recognizes the improvements provided by administration of such acombination of triterpenes as compared to administration of theindividual triterpenes or administration of other combinations oftriterpenes.

SUMMARY OF THE INVENTION

The present inventors have discovered that the clinical benefit providedby compositions containing two or more triterpenes, salts thereof,derivatives thereof and/or prodrugs thereof can be improved to reduceadverse events and improve efficacy. Said improvement is achieved bycontrolling the molar ratio of the triterpenes relative to one another.The invention provides an improved composition comprising a combinationof two or more triterpenes or three or more triterpenes, wherein themolar ratio of the triterpenes is as described herein. The instanttriterpene-based composition, with the specified molar ratio oftriterpenes, exhibits reduced cellular toxicity and increased efficacyas compared to the respective individual triterpenes on atotal-triterpene equimolar basis.

It is an object of the invention to provide an improved triterpene-basedcomposition comprising plural triterpenes as the active ingredientsthereof, wherein the composition provides increased ARE gene expression,and reduced cellular toxicity as compared to other closely relatedtriterpene-based compositions on an equimolar basis.

It is another object of the invention to provide an improvedtriterpene-based composition that provides a balanced expression of AREgenes to provide clinical benefit over a wide dosing range withoutresulting in excessive cellular toxicity.

It is another object of the invention to provide an improvedtriterpene-based composition that provides a broader dose response curveand a broader (wider) therapeutic window as compared to other closelyrelated triterpene-based compositions on a total equimolar basis. Theimproved composition provides a broader therapeutic window, meaning awider dosing range along with lower toxicity especially at the upperlimits of the dosing range, as compared to other closely relatedtriterpene-based compositions on an equimolar basis.

The composition of the invention provides a greater than additiveclinical benefit, meaning a synergistic clinical benefit, e.g.synergistic efficacy, as compared to the individual triterpenes.

The invention provides an improved method of treating a condition,disease or disorder that is therapeutically responsive to triterpene(free base thereof, salt(s) thereof, derivative(s) thereof, and/orprodrug(s) thereof) and/or metabolite(s) thereof, the method comprisingadministering to a subject in need thereof a composition comprising atleast three triterpenes, wherein the molar ratio of the triterpenes isas described herein. Said condition, disease or disorder can beidentified according to the methods described herein or according tomethods known in the art for determining, modeling or predictingtherapeutic responsiveness. The invention also includes methods ofpreventing said condition, disease or disorder. The invention excludesconditions, diseases or disorders that are not therapeuticallyresponsive to said triterpenes or metabolite(s) thereof.

An important clinical benefit provided by the improved composition is asubstantially expanded therapeutic window as compared to the individualtriterpenes administered on a total-triterpene equimolar basis.

Another important clinical benefit provided by the improved compositionis substantially improved efficacy as compared to the individualtriterpenes administered on a total-triterpene equimolar basis.

Another important clinical benefit provided by the improved compositionis substantially reduced adverse events as compared to the individualtriterpenes administered on a total-triterpene equimolar basis.

Embodiments of the invention includes those wherein the condition,disease or disorder is selected from the group consisting of: a)autoimmune condition, disease or disorder; b) neurological condition,disease or disorder; c) anti-inflammatory response-related condition,disease or disorder; d) microbial infection; e) viral infection; f)bacterial infection; g) musculoskeletal condition, disease or disorder;h) excessive cell proliferation related condition, disease or disorder;i) protozoal infection; j) oxidative stress-related condition, diseaseor disorder; k) gastrointestinal condition, disease or disorder; l)angiogenesis-related condition, disease or disorder; m)cyclooxygenase-related condition, disease or disorder; n) cardiovascularcondition, disease or disorder; o) hepatic condition, disease ordisorder; p) age-related condition, disease or disorder; q) bone-relatedcondition, disease or disorder; r) dermatological condition, disease ordisorder; s) parasitic infection; t) renal condition, disease ordisorder; u) metabolic condition, disease or disorder; v)gastrointestinal condition, disease or disorder; and w) pulmonarycondition, disease or disorder.

Embodiments of the invention includes those wherein: a) the autoimmunecondition, disease or disorder is selected from the group consisting ofalopecia areata, celiac disease, diabetes mellitus type 1, Graves'disease, inflammatory bowel disease, multiple sclerosis, psoriasis,rheumatoid arthritis, and systemic lupus erythematosus; b) theneurological condition, disease or disorder as defined herein; c) theviral infection is selected from the group consisting of HSV-1 strain1C, influenza A H7N1, ECHO 6, HIV-1, HEP C, HCV H strain NS5B, HSV-1,HSV-2, ADV-3, ADV-8, ADV-11, HEP B, ENTV CVB1, ENTV EV71, viralhemorrhagic fever (VHF), Arenaviridae infection, Bunyaviridae infection,Filoviridae infection, Flaviviridae infection, Paramyxoviridaeinfection, Togaviridae infection, Filovirus infection, Flavivirusinfection, Henipavirus infection, alphavirus infection, Togavirusinfection, Ebolavirus, Marburgvirus, Alphavirus, Flavivirus, YellowFever, Dengue Fever, Japanese Enchephalitis, West Nile Viruses,Zikavirus, Venezuelan Equine Encephalomyelitis (encephalitis) (VEE)virus, Chikungunya virus, Western Equine Encephalomyelitis(encephalitis) (WEE) virus, Eastern Equine Encephalomyelitis(encephalitis) (EEE) virus, Tick-borne Encephalitis, Kyasanur ForestDisease, Alkhurma Disease, Omsk Hemorrhagic Fever, Hendra virus, Nipahvirus, and species thereof, d) the bacterial infection is selected fromthe group consisting of Aeromonas caveae infection, Bacillus cereusinfection, Bacillus sphaericus infection, Bacillus subtilis infection,Enterococcus faecalis infection, Escherichia coli infection, Klebisiellapneumoniae infection, Listeria monocytogenes infection, Mycobacteriumtuberculosis infection, Pseudomonas aeruginosa infection, Pseudomonassyrinagae infection, Ralstonia solanacearum infection, Shigella flexneriinfection, Staphylococcus aureus infection, Staphylococcus epidermisinfection, Streptococcus mutans infection, Streptococcus pneumoniaeinfection, Streptococcus sobrinus infection, Streptomyces scabiesinfection, Vibrio cholerae infection, vancomycin-resistant enterococciinfection, methicillin-resistant Staphylococcus aureus infection, andproteobacteria infection; e) the musculoskeletal condition, disease ordisorder is selected from the group consisting of skeletal muscleatrophy, muscular atrophy, muscular dysfunction, amyotrophic lateralsclerosis, and sarcopenia; f) the excessive cell proliferation relatedcondition, disease or disorder is selected from the group consisting ofcancer, tumor, colorectal cancer, head and neck cancer, adrenal corticalcancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, bonemetastasis, sarcomas of bone, brain cancer, breast cancer, cervicalcancer, non-Hodgkin's lymphoma, rectal cancer, esophageal cancer, eyecancer, gallbladder cancer, gastrointestinal carcinoid tumor,gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma,kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lungcancer (both non-small cell and small cell carcinomas), lung carcinoidtumors, malignant mesothelioma, metastatic cancer, multiple myeloma,myelodysplastic syndrome, nasal cavity and paranasal cancer,nasopharyngeal cancer, neuroblastoma, neoplasms of the central nervoussystem, oral cavity and oropharyngeal cancer, osteosarcoma, ovariancancer, pancreatic cancer, penile cancer, pituitary cancer, prostatecancer, retinoblastoma, salivary gland cancer, sarcoma, skin cancer,stomach cancer, testicular cancer, thymus cancer, thyroid cancer, cancerof the ureter; uterine sarcoma, vaginal cancer, vulva cancer or Wilm'stumor; g) the protozoal infection is selected from the group consistingof Leishmania amazonensis infection, Plasmodium falciparum infection,Trypanosoma brucei rhodesiense infection, Trypanosoma cruzi infection,and malaria; h) the cardiovascular condition, disease or disorder isselected from the group consisting of myocardial infarction, stroke,atherosclerosis, hypertension, varicose veins, and damage caused byC-reactive protein; i) the hepatic condition, disease or disorder isselected from the group consisting of hepatic lipid accumulation,hepatic steatosis, hepatic fibrosis, and hepatic degeneration(degradation); j) the bone-related condition, disease or disorder isselected from the group consisting of osteoporosis, and particle inducedosteolysis; k) the renal condition, disease or disorder is selected fromthe group consisting of nephrotic syndrome, and focal segmentalglomerulosclerosis; or 1) the pulmonary condition, disease or disorderis selected from the group consisting of acute lung injury, chronicobstructive pulmonary disorder, and asthma.

In some embodiments, the neurological condition is selected from thegroup consisting of neurological disease, neurological disorder,tauopathy, and stroke. In some embodiments, the neurological disease isa neurodegenerative disease. In some embodiments, the neurodegenerativedisease is selected from the group consisting of Huntington's disease,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,bovine spongiform encephalopathy, multiple sclerosis, diabeticneuropathy, autism and juvenile neuronal ceroid lipofuscinosis. In someembodiments, stroke is stroke-mediated ischemic injury. In someembodiments, the neurological condition is a tauopathy, which is aneurodegenerative disease having an etiology associated with animbalance in the Tau3R/Tau4R ratio in a subject. Tauopathies are a classof neurodegenerative diseases resulting from the pathologicalaggregation of tau proteins in the human brain. In some embodiments, thetauopathy is Down's syndrome, Pick's disease, corticobasal degeneration,some variants of prions disease, Alzheimer's disease, progressivesupranuclear palsy or frontotemporal dementia. The individual steps ofthe methods of the invention can be conducted at separate facilities orwithin the same facility.

The invention provides an improved method of treating atriterpene-responsive condition, disease or disorder comprisingadministering to a subject in need thereof an improved compositioncomprising (consisting essentially of) at least three triterpenes. Themolar ratio of triterpenes has been found to impact efficacy andtoxicity/safety of the composition(s). Embodiments of the inventioninclude those wherein the molar ratio of triterpenes is as describedherein. The molar ratio of triterpenes in the improved composition isdifferent than and improved over that found in PBI-05204 and PBI-04711.

In some embodiments, the composition contains triterpenes as the solepharmacologically active ingredients (agents). The composition canexclude steroid, cardiac glycoside, biologically/pharmacologicallyactive polysaccharide, and/or non-cardiac glycoside steroid.

In one aspect, the invention provides a method of treating, in a subjectin need thereof, a triterpene-responsive condition, disease or disorderwith a composition comprising at least two or at least three triterpenespresent at a molar ratio as described herein, the method comprising:

-   determining that the subject has a triterpene-responsive condition,    disease or disorder; and-   indicating administration of a therapeutically effective amount of    said composition to the subject.

The invention also provides a method of treating, in a subject in needthereof, a condition, disease or disorder that is therapeuticallyresponsive to triterpene and/or metabolite thereof with atriterpene-based composition, the method comprising administering to thesubject a therapeutically effective amount of said triterpene-basedcomposition, wherein the molar ratio of triterpenes in said compositionare as described herein.

In one aspect, the invention provides a method of treating, in a subjectin need thereof, a neurological condition, disease or disorder with aneuroprotective composition comprising at least two or at least threetriterpenes, the method comprising:

-   determining that the subject has a neurological condition, disease    or disorder; and-   indicating administration of a therapeutically effective amount of    the neuroprotective composition to the subject.

The invention also provides a method of treating, in a subject in needthereof, a neurological disease or disorder with a neuroprotectivecomposition, the method comprising administering to the subject atherapeutically effective amount of the neuroprotective composition.

Some embodiments of the invention include those wherein: 1) the subjectis prescribed and administered a therapeutically relevant dose of thecomposition; 2) the subject is administered the composition according toa prescribed dosing regimen; 3) the composition excludes cardiacglycoside; 4) the composition excludes a therapeutically effectiveamount of cardiac glycoside; 5) the composition excludes oleandrin; 6)the composition excludes a neriifolin; 7) the composition excludes apharmacologically active polysaccharide obtained from Nerium species orThevetia species; 8) the composition comprises a synthetic mixture of atleast two or at least three triterpenes present at a molar ratio asdescribed herein; or 9) a combination of any two or more of the above.

The invention also provides a method of treating a neurologicalcondition in a subject in need thereof comprising:

-   determining whether or not the neurological condition in the subject    is Alzheimer's disease, Huntington's disease, stroke, Parkinson's    disease or other neurological condition;-   indicating administration of a neuroprotective composition;-   administering an initial dose of the neuroprotective composition to    the subject according to a prescribed initial dosing regimen for a    period of time;-   periodically determining the adequacy of the subject's clinical    response and/or therapeutic response to treatment with the    neuroprotective composition; and-   if the subject's clinical response and/or therapeutic response is    adequate, then continuing treatment with the neuroprotective    composition as needed until the desired clinical endpoint is    achieved; or-   if the subject's clinical response and/or therapeutic response are    inadequate at the initial dose and initial dosing regimen, then    escalating or deescalating the dose of the neuroprotective    composition until the desired clinical response and/or therapeutic    response in the subject is achieved.

The invention also provides a method of preventing or reducing theincidence of occurrence of a triterpene-responsive condition, disease ordisorder in a population of subjects at risk thereof, the methodcomprising:

-   administering an effective dose of a triterpene-based composition on    a recurring basis for an extended period of time to one or more    subjects in said population of subjects, thereby preventing or    reducing the incidence of said condition, disease or disorder in the    population; wherein-   the composition comprises at least two or at least three triterpenes    selected from the group consisting of oleanolic acid, ursolic acid,    and betulinic acid, each of said triterpenes being present in a form    which is independently selected upon each occurrence from the group    consisting of free acid form, salt form, derivative form and prodrug    form, and the molar ratio of said triterpenes being as described    herein.

The invention also provides a method of preventing or reducing theincidence of occurrence of a neurological condition in a population ofsubjects at risk thereof, the method comprising:

-   administering an effective dose of neuroprotective composition on a    recurring basis for an extended period of time to one or more    subjects in a population of subjects at risk of suffering from a    neurological condition such as Alzheimer's disease, Huntington's    disease, Parkinson's disease, stroke or other neurological    condition, thereby preventing or reducing the incidence of the    neurological condition in the population; wherein-   said neuroprotective composition comprises at least two or at least    three triterpenes selected from the group consisting of oleanolic    acid, ursolic acid, and betulinic acid, each of said triterpenes    being present in a form which is independently selected upon each    occurrence from the group consisting of free acid form, salt form,    derivative form and/or prodrug form, and the molar ratio of said    triterpenes being as described herein.

The invention also includes embodiments wherein: a) the method furthercomprises indicating administration of the neuroprotective compositionto the one or more subjects; b) the method further comprisesadministering an effective dose of the neuroprotective composition tothe subject according to a prescribed dosing regimen for a period oftime; c) the method further comprises periodically determining theadequacy of one or more subject's clinical response and/or therapeuticresponse to treatment with the neuroprotective composition; d) if thesubject's clinical response and/or therapeutic response is adequate,then the method further comprises continuing treatment with theneuroprotective composition as needed until the desired clinicalendpoint is achieved; e) if the subject's clinical response and/ortherapeutic response are inadequate at the initial dose and initialdosing regimen, then the method further comprises escalating ordeescalating the dose of neuroprotective composition until the desiredclinical response and/or therapeutic response in the subject isachieved; f) the neuroprotective composition is administered to pluralsubjects in a population; g) the recurring basis is daily, every otherday, every second day, every third day, every fourth day, every fifthday, every sixth day, weekly, every other week, every second week, everythird week, monthly, bimonthly, semi-monthly, every other month everysecond month, quarterly, every other quarter, trimesterly, seasonally,semi-annually and/or annually; h) the extended period is one or moreweeks, one or more months, one or more quarters and/or one or moreyears; i) the effective dose is administered one or more times in a day;j) the method further comprises identifying a population of subjects atrisk of suffering from a neurological condition such as Alzheimer'sdisease, Huntington's disease, Parkinson's disease, stroke or otherneurological condition; k) the population of subjects at risk ischaracterized by advancing age of the subject, familial history of theneurological condition, genetic predisposition to occurrence ofneurological condition, the presence and expression of ApoE4 gene in thesubject, female gender (twice as many women get Alzheimer's disease thanmen), cardiovascular disease (e.g. high blood pressure and highcholesterol levels), diabetes (especially Type 2 or adult onset forms ofthis disease), Down's Syndrome, head injury, low levels of formaleducation, smoking, excessive alcohol consumption and/or drug abuse;or 1) a combination thereof.

The invention also provides a time-delayed method of treating stroke ina subject comprising:

-   within a delay period after a subject has suffered the stroke,    administering an initial dose of neuroprotective composition    according to an initial dosing regimen;-   determining the adequacy of subject's clinical response and/or    therapeutic response to treatment with the neuroprotective    composition; and-   if the subject's clinical response and/or therapeutic response is    adequate, then continuing treatment with neuroprotective composition    as needed until the desired clinical endpoint is achieved; or-   if the subject's clinical response and/or therapeutic response are    inadequate at the initial dose and initial dosing regimen, then    escalating or deescalating the dose of neuroprotective composition    until the desired clinical response and/or therapeutic response in    the subject is achieved.

Some embodiments of the invention include those wherein: 1) the delayperiod is 10 hours or less, 8 hours or less, 6 hours or less, 4 hours orless, 3 hours or less, 2 hours or less, 1 hour or less, 45 minutes orless, 30 minutes or less, 20 minutes or less or 10 min or less; 2)determining the adequacy of a subject's clinical and/or therapeuticresponse is done by assessments of any weakness of the face, arm and/orleg on one side of the body, numbness in the face, arm, and/or leg onone side of the body, inability to understand spoken language, inabilityto speak or speak clearly, inability to write, vertigo and/or gaitimbalance, double vision and an unusually severe headache; or 3) acombination thereof.

The invention also provides use of a triterpene-based composition in themanufacture of a medicament for the treatment of a triterpene-responsivecondition, disease or disorder in a subject. In some embodiments, themanufacture of such a medicament comprises: providing said composition;including a dose of said composition in a pharmaceutical dosage form;and packaging the pharmaceutical dosage form.

The invention also provides a pharmaceutical composition comprising saidcomposition for the treatment of a condition, disease or disorder in asubject. The manufacture can also include one or more additional stepssuch as: delivering the packaged dosage form to a vendor (retailer,wholesaler and/or distributor); selling or otherwise providing thepackaged dosage form to a subject having a neurological condition;including with the medicament a label and a package insert, whichprovides instructions on use, dosing regimen, administration, contentand toxicology profile of the dosage form.

In some embodiments, the treatment of a triterpene-responsive condition,disease or disorder comprises: determining that a subject has atriterpene-responsive condition, disease or disorder; indicatingadministration of said composition to the subject according to a dosingregimen; administering to the subject one or more pharmaceutical dosageforms containing said composition, wherein the one or morepharmaceutical dosage forms is administered according to the dosingregimen.

In some embodiments, the subject having a neurological condition, i.e.the subject in need thereof, is part of a population of such subjects.The invention provides a method for improving the clinical status of astatistically significant number of subjects of in a population ofsubjects having a neurological condition, the method comprising:administering to the population of subjects a neuroprotectivecomposition as described herein; and determining the clinical status ofthe subjects. In some embodiments, the statistically significant numberis at least 5% of the population.

In some embodiments, the neurological condition is Alzheimer's disease,Huntington's disease, stroke, Parkinson's disease, a tauopathy or otherneurological condition, such as described herein.

Treatment of the subject with said composition is continued as needed.The dose or dosing regimen can be adjusted as needed until the patientreaches the desired clinical endpoint(s) such as a reduction oralleviation of specific symptoms associated with the condition, diseaseor disorder being treated. Determination of the adequacy of clinicalresponse and/or therapeutic response can be conducted by a clinicianfamiliar with the condition, disease or disorder being treated.

Embodiments of the invention also include a triterpene-based improvedcomposition comprising at least two or at least three triterpenes,wherein: said triterpenes are present at a molar ratio as describedherein; and said triterpenes are selected from the group consisting ofoleanolic acid, ursolic acid, and betulinic acid, each of saidtriterpenes being present in a form which is independently selected uponeach occurrence from the group consisting of free acid form, salt form,derivative form, and prodrug form.

In some embodiments, the invention provides a triterpene-based improvedcomposition exhibiting therapeutic activity as described herein whenadministered to a subject. In some embodiments, the methods of theinvention employ said composition as described herein.

In each embodiment of the invention, a triterpene is independently uponeach occurrence present in native form (unmodified free acid, unmodifiedsalt or combination thereof form), derivative form, prodrug form, or acombination of two or more of said forms. For example, a triterpene canbe present as a mixture of free acid form and salt form, or a mixture offree acid form and derivative form, or a mixture of free acid form andprodrug form, or a mixture of two or more forms, or a mixture of threeor more forms. The term “triterpene” as used herein refers to each formor any of said forms or any mixture of said forms. The table belowspecifies various combinations of triterpene forms.

Triterpene Form Present (Y/N) Sample Free acid Salt Derivative ProdrugCombination 1 Y Y N N Combination 2 Y N Y N Combination 3 Y N N YCombination 4 N Y Y N Combination 5 N Y N Y Combination 6 N N Y YCombination 7 N Y N Y Combination 8 Y Y Y N Combination 9 Y N Y YCombination 10 Y Y N Y Combination 11 N Y Y Y Combination 12 Y Y Y Y

Embodiments of the invention include those wherein the triterpene-basedimproved composition is a neuroprotective composition comprising(consisting essentially of) at least two triterpenes present at a molarratio as described herein. In some embodiments, the neuroprotectivecomposition of the invention comprises (consists essentially of) atleast three triterpenes present at a molar ratio as described herein.

In some embodiments, the composition of the invention comprises(consists essentially of) oleanolic acid (free acid, salt, derivative,and/or prodrug thereof) and ursolic acid (free acid, salt, derivative,and/or prodrug thereof) and optionally at least one other triterpene,wherein the molar ratio of triterpenes is as described herein. Forexample, the composition can further comprise betulinic acid (free acid,salt, derivative, and/or prodrug thereof) or at least one othertriterpene.

In some embodiments, the composition of the invention comprises(consists essentially of) oleanolic acid (free acid, salt, derivative,and/or prodrug thereof) and betulinic acid (free acid, salt, derivative,and/or prodrug thereof) and optionally at least one other triterpene,wherein the molar ratio of triterpenes is as described herein. Forexample, the composition can further comprise ursolic acid (free acid,salt, derivative, and/or prodrug thereof) or at least one othertriterpene.

Some embodiments of the invention provide a composition comprising(consisting essentially of) at least oleanolic acid (free acid, salt,derivative, and/or prodrug thereof), betulinic acid (free acid, salt,derivative, and/or prodrug thereof), and ursolic acid (free acid, salt,derivative, and/or prodrug thereof), wherein the molar ratio of saidtriterpenes is as described herein.

Some embodiments of the invention provide an improved compositioncomprising (consisting essentially of) at least: oleanolic acid (presentas free acid and/or salt thereof), betulinic acid (present as free acidand/or salt thereof), and ursolic acid (present as free acid and/or saltthereof), wherein the molar ratio of said compounds is as describedherein.

A pharmaceutical dosage form comprises the composition and one or morepharmaceutically acceptable excipients.

In some embodiments, the composition comprises (consists essentially of)the triterpenes oleanolic acid, ursolic acid, and betulinic acid,wherein the molar ratio of triterpenes is as described herein. In someembodiments, the composition is included in a pharmaceutical compositionfurther comprising at least one pharmaceutically acceptable excipient.

In some embodiments, the majority of pharmacologically active componentin the composition is oleanolic acid. Oleanolic acid can be present inmolar excess over ursolic acid and/or over betulinic acid. Oleanolicacid can be present in molar excess over the combined total moles ofursolic acid and betulinic acid. Oleanolic acid and ursolic acid cantogether (sum total) or individually be present in molar excess overbetulinic acid. Oleanolic acid and betulinic acid can together (sumtotal) or individually be present in molar excess over ursolic acid.Ursolic acid and betulinic acid can be present at about the samecontent. Ursolic acid can be present in molar excess over betulinicacid. Betulinic acid can be present in molar excess over ursolic acid.

Embodiments of the invention include those wherein: a) oleanolic acid ispresent in molar excess over the combined total moles of ursolic acidand betulinic acid, and ursolic acid and betulinic acid are present atabout the same molar content; b) oleanolic acid is present in molarexcess over the combined total moles of ursolic acid and betulinic acid,and ursolic acid is present in molar excess over betulinic acid; c)oleanolic acid is present in molar excess over the combined total molesof ursolic acid and betulinic acid, and betulinic acid is present inmolar excess over ursolic acid; d) oleanolic acid is present in molarexcess over ursolic acid, and ursolic acid is present in molar excessover betulinic acid; or e) oleanolic acid is present in molar excessover betulinic acid, and betulinic acid is present in molar excess overursolic acid.

When oleanolic acid (free acid, salt, derivative, and/or prodrugthereof), ursolic acid (free acid, salt, derivative, and/or prodrugthereof) and betulinic acid (free acid, salt, derivative, and/or prodrugthereof) are present as the primary or sole triterpenes, the molar ratioof oleanolic acid (O):ursolic acid (U):betulinic acid (B) is about10:about 1:about 1, about 9-11:about 0.5-1.5:about 0.5-1.5, about9.5-10.5:about 0.75-1.25:about 0.75-1.25, about 9.5-10.5:about0.8-1.2:about 0.8-1.2, about 9.75-10.5:about 0.9-1.1:about 0.9-1.1,about 9-12:about 0.15-2.5:about 0.15-2.5, about 9-12:about 0.2-2.5:about0.2-2.5, about 9-12:about 0.25-2.5:about 0.25-2.5, about 9-12:about0.35-2.5:about 0.35-2.5, about 9-12:about 0.45-2.5:about 0.45-2.5, about9-12:about 0.5-5:about 0.5-2.5, about 9-12:about 0.16-2:about 0.16-2,about 9-12:about 0.2-2:about 0.2-2, about 9-12:about 0.25-2:about0.25-2, about 9-12:about 0.25-2:about 0.25-2, about 9-12:about0.45-2:about 0.45-2, about 9-12:about 0.5-2:about 0.5-2, about9-12:about 0.16-1.5:about 0.16-1.5, about 9-12:about 0.2-1.5:about0.2-1.5, about 9-12:about 0.25-1.5:about 0.25-1.5, about 9-12:about0.7-1.5:about 0.35-1.5, about 9-12:about 0.45-1.5:about 0.45-1.5, about9-12:about 0.5-1.5:about 0.5-1.5, about 9-12:about 0.16-1:about 0.16-1,about 9-12:about 0.2-1:about 0.2-1, about 9-12:about 0.25-1:about0.25-1, about 9-12:about 0.35-1:about 0.35-1, about 9-12:about0.45-1:about 0.45-1, about 9-12:about 0.5-1:about 0.5-1, about 10:about0.5-2.5:about 0.5-2.5, about 10:about 0.1-1.5:about 0.1-1.5, about9-12:about 0.25-0.75:about 0.25-0.75, about 9.5-10.5:about0.35-0.7:about 0.35-0.7, about 9.5-10.5:about 0.4-0.6:about 0.4-0.6, orabout 9.75-10.5:about 0.45-0.6:about 0.45-0.6.

When oleanolic acid (free acid, salt, derivative, and/or prodrugthereof) and ursolic acid (free acid, salt, derivative, and/or prodrugthereof) are present as the primary or sole triterpenes, the molar ratioof oleanolic acid:ursolic acid is about 9-12:about 0.33-5, about9-12:about 0.4-5, about 9-12:about 0.5-5, about 9-12:about 0.7-5, about9-12:about 0.9-5, about 9-12:about 1-5, about 9-12:about 0.33-4, about9-12:about 0.4-4, about 9-12:about 0.5-4, about 9-12:about 0.7-4, about9-12:about 0.9-4, about 9-12:about 1-4, about 9-12:about 0.33-3, about9-12:about 0.4-3, about 9-12:about 0.5-3, about 9-12:about 0.7-3, about9-12:about 0.9-3, about 9-12:about 1-3, about 9-12:about 0.33-2, about9-12:about 0.4-2, about 9-12:about 0.5-2, about 9-12:about 0.7-2, about9-12:about 0.9-2, about 9-12:about 1-2, about 10:about 1-5, about10:about 1-3, about 9-12:about 0.5-1.5, about 9-11:about 0.5-1.5, about9.5-10.5:about 0.75-1.25, about 9.5-10.5:about 0.8-1.2, or about9.75-10.5:about 0.9-1.1.

When oleanolic acid (free acid, salt, derivative, and/or prodrugthereof) and betulinic acid (free acid, salt, derivative, and/or prodrugthereof) are present as the primary or sole triterpenes, the molar ratioof oleanolic acid:betulinic acid is about 9-12:about 0.33-5, about9-12:about 0.4-5, about 9-12:about 0.5-5, about 9-12:about 0.7-5, about9-12:about 0.9-5, about 9-12:about 1-5, about 9-12:about 0.33-4, about9-12:about 0.4-4, about 9-12:about 0.5-4, about 9-12:about 0.7-4, about9-12:about 0.9-4, about 9-12:about 1-4, about 9-12:about 0.33-3, about9-12:about 0.4-3, about 9-12:about 0.5-3, about 9-12:about 0.7-3, about9-12:about 0.9-3, about 9-12:about 1-3, about 9-12:about 0.33-2, about9-12:about 0.4-2, about 9-12:about 0.5-2, about 9-12:about 0.7-2, about9-12:about 0.9-2, about 9-12:about 1-2, about 10:about 1-5, about10:about 1-3, about 9-12:about 0.5-1.5, about 9-11:about 0.5-1.5, about9.5-10.5:about 0.75-1.25, about 9.5-10.5:about 0.8-1.2, or about9.75-10.5:about 0.9-1.1.

The individual steps of the methods of the invention can be conducted atseparate facilities or within the same facility. Any of the methods ofthe invention described herein can be used in combination with any ofthe compositions of the invention described herein.

The invention includes all combinations of the aspects, embodiments andsub-embodiments of the invention disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

The following figures form part of the present description and describesome of the prior art and exemplary embodiments of the claimedinvention. The skilled artisan will, in light of these figures and thedescription herein, be able to practice the invention without undueexperimentation.

FIG. 1 (prior art; Van Kanegan: vide supra) depicts the results of thecomparative evaluation of PBI-05204 (PBI) and PBI-04711 (Fraction 0-4 ofPBI-05204) in a brain slice oxygen-glucose deprivation (OGD) assay.Coronal brain slice explants were prepared and subjected to 5.5 mintransient OGD. Numbers of healthy cortical pyramidal neurons in eachbrain slice were scored 24 h later. The first 3 bars in the graph show:control brain slices not subjected to OGD (“Control”); negative-controlbrain slices subjected to OGD and treated with DMSO carrier only(“OGD”); and positive-control brain slices subjected to OGD and treatedwith 23 μg/ml of the full PBI-05204 extract (“PBI 23”). The fraction wastested at the concentrations indicated in units of g/ml. Fraction 0-4provided significant neuroprotection at the concentrations tested(concentrations of Fraction 0-3 of 10 g/ml and above exhibited toxicity;data not shown).

FIGS. 2A-2D (prior art: Van Kanegan: vide supra) depict the results ofARE gene expression assays for Fraction 0-4 (PBI-04711): a) Gclcexpression (FIG. 2A); b) Nqo1 expression (FIG. 2B); c) Srx expression(FIG. 2C); and d) Hmox1 expression (FIG. 2D). Primary mousecorticostriatal co-cultures were treated with Fraction 0-4 at theconcentrations indicated for 6 h, then harvested and processed for qPCRanalysis of the ARE target genes shown. Quantitative RNA values arenormalized to the GAPDH reference control and fold-expression changesare expressed relative to the DMSO-carrier only condition (“0”) set to avalue of 1.

FIG. 3 (prior art: Van Kanegan: vide supra) depicts the results of AREgene expression assays for Fraction 0-4 and the individual triterpenesoleanolic acid, ursolic acid, betulinic acid and uvaol (also referred toas uvalol). “X” symbols denote concentrations of compounds which inducedtoxicity and for which recovery of residual mRNA was insufficient tosupport qPCR analysis. Rat primary corticostriatal co-cultures weretreated for 6 h with Fraction 0-4 (in μg/ml) or oleanolic acid, ursolicacid, betulinic acid, or uvaol (all in μM) at the concentrationsindicated, then harvested and processed for qPCR analysis of the AREtarget genes shown. Quantitative RNA values were normalized to the GAPDHreference control and fold-expression changes are expressed relative tothe DMSO-carrier only condition (“--”) set to a value of 1. Dark barsdenote statistically significant differences with respect to theDMSO-carrier only control by a Student's t-test at p<0.05.

FIG. 4 depicts the results of expression assays for Fraction 0-4 and theindividual triterpenes ursolic acid and betulinic acid at moreclosely-spaced concentration ranges. Rat primary corticostriatalco-cultures were treated for 6 h with Fraction 0-4 (in μg/ml) or ursolicacid and betulinic acid (in μM) at the concentrations indicated, thenharvested and processed for qPCR analysis of the ARE target genes shown.Quantitative RNA values were normalized to the GAPDH reference controland fold-expression changes are expressed relative to the DMSO-carrieronly condition (“--”) set to a value of 1. Dark blue bars denotestatistically significant differences with respect to the DMSO-carrieronly control by a Student's t-test at p<0.05. Betulinic acid, likeursolic acid, is also able to induce clear upregulation of Srx and Hmox1despite its toxicity at higher concentrations.

FIG. 5 (prior art: Van Kanegan: vide supra) depicts the results ofcomparative evaluation in a neuroprotection OGD assay of oleanolic acid(OA), ursolic acid (UA), betulinic acid (BA) and uvaol (Uva).Concentration-response relations for UA, BA, and Uva (all in μg/ml) inthe brain slice OGD assay are shown. Averages for 2 independentexperiments are included for each compound, with the OGDnegative-control condition scaled to 100% and data plotted on the sameaxes for ease of comparison. The positive control was 4 μg/ml oleanolicacid (O). Note that these are equimolar concentrations for each compoundas the molecular weights for all are identical except for uvaol whichwas tested at 0.039, 0.39, and 3.88 μg/ml rounded to a singlesignificant digit for display purposes. Dark bars denote statisticallysignificant differences with respect to the OGD negative control byANOVA followed by Dunnett's post hoc comparison test at the 0.05confidence level.

FIGS. 6A, 6B, 7A, 7B, 8A, and 8B depict the results of comparativecellular toxicities of the triterpenes to primary corticalstriatalneuronal co-cultures containing glia evaluated according to the examplebelow: ursolic acid (FIGS. 6A, 6B), betulinic acid (FIGS. 7A, 7B) andoleanolic acid (FIGS. 8A, 8B). For FIGS. 6A, 7A and 8A, propidium iodidewas added for one hour then the numbers of PI-positive cells were scoredunder automated high-content analysis on the Cellomics ArrayScan VTI.For FIGS. 6B, 7B and 8B, MTS substrate was added for two hours thenco-culture wells were measured for absorbance at 450 nm using amulti-well plate reader.

FIGS. 9A-9C depict the results of comparative neuroprotection oftriterpene mixtures (Composition I: molar ratio of O:U:B is 3:2.2:1 asin PBI-04711 (Fxn 0-4; FIG. 9A); Composition II: molar ratio of O:U:B is7.8:7.4:1 as in PBI-05204 (FIG. 9B); and Composition III: molar ratio ofO:U:B is about 10:1:1 as per the improved composition of the invention(PBI-01011; FIG. 9C)) determined in a brain slice assay for ischemicstroke according to Example 3. Numbers of healthy cortical pyramidalneurons per brain slice are shown relative to the negative controlcondition set to 100% (second bar in each graph) of brain slices exposedto oxygen-glucose deprivation (OGD) and vehicle (DMSO) only. Values forpositive control brain slices not exposed to OGD are shown in the firstbar of each graph. Mean values+SEM are shown averaged over 3-5independent runs for each triterpene mixture; light blue bars denotestatistically significant differences with respect to the OGD negativecontrol by ANOVA followed by Dunnett's post hoc comparison test atp<0.05.

FIGS. 10A and 10B depict the results of comparative Srx (FIG. 10A) andHmox1 (FIG. 10B) expression assays for PBI-05204, PBI-04711 (alsoreferred to as Fxn 0-4), oleanolic acid (O), ursolic acid (U), betulinicacid (B) and combinations of the triterpenes present at the specifiedmolar ratios. Quantitative RNA values were normalized to the GAPDHreference control and fold-expression changes are shown relative to theDMSO-carrier only condition set to a value of 1. Dark blue bars denotestatistically significant differences with respect to the DMSO-carrieronly control by a Student's t-test at p<0.05. Striped, red bars denoteconditions which induced excessive levels of ARE gene expression,namely, by more than 10-fold.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the individually named triterpenes can independently beselected upon each occurrence in their native (unmodified) free acidform, native salt form, derivative form, prodrug form, or a combinationthereof. Compositions containing and methods employing deuterated formsof the triterpenes are also within the scope of the invention. The totalmoles of a triterpene present in a composition is the sum total of themoles of each form of said triterpene. For example, the total moles of“oleanolic acid” present in a composition is the sum total of moles ofnative free acid form+native salt form(s)+derivative form(s)+prodrugform(s).

Oleanolic acid derivatives, prodrugs and salts are disclosed in US20150011627 A1 to Gribble et al. which published Jan. 8, 2015, US20140343108 A1 to Rong et al which published Nov. 20, 2014, US20140343064 A1 to Xu et al. which published Nov. 20, 2014, US20140179928 A1 to Anderson et al. which published Jun. 26, 2014, US20140100227 A1 to Bender et al. which published Apr. 10, 2014, US20140088188 A1 to Jiang et al. which published Mar. 27, 2014, US20140088163 A1 to Jiang et al. which published Mar. 27, 2014, US20140066408 A1 to Jiang et al. which published Mar. 6, 2014, US20130317007 A1 to Anderson et al. which published Nov. 28, 2013, US20130303607 A1 to Gribble et al. which published Nov. 14, 2013, US20120245374 to Anderson et al. which published Sep. 27, 2012, US20120238767 A1 to Jiang et al. which published Sep. 20, 2012, US20120237629 A1 to Shode et al. which published Sep. 20, 2012, US20120214814 A1 to Anderson et al. which published Aug. 23, 2012, US20120165279 A1 to Lee et al. which published Jun. 28, 2012, US20110294752 A1 to Arntzen et al. which published Dec. 1, 2011, US20110091398 A1 to Majeed et al. which published Apr. 21, 2011, US20100189824 A1 to Arntzen et al. which published Jul. 29, 2010, US20100048911 A1 to Jiang et al. which published Feb. 25, 2010, and US20060073222 A1 to Arntzen et al. which published Apr. 6, 2006, theentire disclosures of which are hereby incorporated by reference.

Ursolic acid derivatives, prodrugs and salts are disclosed in US20150011627 A1 to Gribble et al. which published Jan. 8, 2015, US20130303607 A1 to Gribble et al. which published Nov. 14, 2013, US20150218206 A1 to Yoon et al. which published Aug. 6, 2015, U.S. Pat.No. 6,824,811 to Fritsche et al. which issued Nov. 30, 2004, U.S. Pat.No. 7,718,635 to Ochiai et al. which issued May 8, 2010, U.S. Pat. No.8,729,055 to Lin et al. which issued May 20, 2014, and U.S. Pat. No.9,120,839 to Yoon et al. which issued Sep. 1, 2015, the entiredisclosures of which are hereby incorporated by reference.

Betulinic acid derivatives, prodrugs and salts are disclosed in US20150011627 A1 to Gribble et al. which published Jan. 8, 2015, US20130303607 A1 to Gribble et al. which published Nov. 14, 2013, US20120237629 A1 to Shode et al. which published Sep. 20, 2012, US20170204133 A1 to Regueiro-Ren et al. which published Jul. 20, 2017, US20170096446 A1 to Nitz et al. which published Apr. 6, 2017, US20150337004 A1 to Parthasaradhi Reddy et al. which published Nov. 26,2015, US 20150119373 A1 to Parthasaradhi Reddy et al. which publishedApr. 30, 2015, US 20140296546 A1 to Yan et al. which published Oct. 2,2014, US 20140243298 A1 to Swidorski et al. which published Aug. 28,2014, US 20140221328 A1 to Parthasaradhi Reddy et al. which publishedAug. 7, 2014, US 20140066416 A1 tp Leunis et al. which published Mar. 6,2014, US 20130065868 A1 to Durst et al. which published Mar. 14, 2013,US 20130029954 A1 to Regueiro-Ren et al. which published Jan. 31, 2013,US 20120302530 A1 to Zhang et al. which published Nov. 29, 2012, US20120214775 A1 to Power et al. which published Aug. 23, 2012, US20120101149 A1 to Honda et al. which published Apr. 26, 2012, US20110224182 to Bullock et al. which published Sep. 15, 2011, US20110313191 A1 to Hemp et al. which published Dec. 22, 2011, US20110224159 A1 to Pichette et al. which published Sep. 15, 2011, US20110218204 to Parthasaradhi Reddy et al. which published Sep. 8, 2011,US 20090203661 A1 to Safe et al. which published Aug. 13, 2009, US20090131714 A1 to Krasutsky et al. which published May 21, 2009, US20090076290 to Krasutsky et al. which published Mar. 19, 2009, US20090068257 A1 to Leunis et al. which published Mar. 12, 2009, US20080293682 to Mukherjee et al. which published Nov. 27, 2008, US20070072835 A1 to Pezzuto et al. which published Mar. 29, 2007, US20060252733 A1 to Jansen et al. which published Nov. 9, 2006, and US2006025274 A1 to O'Neill et al. which published Nov. 9, 2006, the entiredisclosures of which are hereby incorporated by reference.

When a derivative or prodrug of a triterpene is used, the moles of eachis determined according to the molar equivalents of the parent free acidtriterpene. For example, one mole of the methyl ester derivative ofoleanolic acid (methyl oleanolate) is the molar equivalent of one moleof oleanolic acid and vice versa.

The invention provides a method of treating a triterpene-responsivecondition, disease or disorder by administration of an effective dose(therapeutically effective dose) of a triterpene-based composition ofthe invention to a subject in need thereof. The composition isadministered according to a dosing regimen best suited for the subject,the suitability of the dose and dosing regimen to be determinedclinically according to conventional clinical practices and clinicaltreatment endpoints for the condition, disease or disorder beingtreated.

In some embodiments, the neurodegenerative disorder or neurologicalcondition being treated has an etiology associated with anover-expression of tau proteins and/or an imbalance in the Tau3R/Tau4Rratio in a subject. Such a condition is termed a tauopathy. Exemplarytauopathies include Down's syndrome, Pick's disease, some variants ofprions disease, Alzheimer's disease, progressive supranuclear palsy orfrontotemporal dementia, corticobasal degeneration, Guam parkinsonismdementia complex, dementia with argyrophilic grains, Niemann-Pickdisease Type C, and dementia pugilistic.

In some embodiments, the neurodegenerative disorder or neurologicalcondition being treated has an etiology associated with abnormal oratypical proteolysis of amyloid beta precursor protein, accumulation ofamyloid beta protein in the synapses of the neurons, formation ofamyloid fibrils in the synapses of the neurons, or formation of amyloidplaques in the synapses of the neurons. Exemplary of such disorders orconditions is Alzheimer's disease. A subject treated according to theinvention will exhibit a therapeutic response.

By “therapeutic response” is meant that a subject suffering from thedisease or disorder will enjoy at least one of the following clinicalbenefits as a result of treatment with the triterpene-based composition:amelioration of the disease or disorder, reduction in the occurrence ofsymptoms associated with the disease or disorder, partial remission ofthe disease or disorder, full remission of the disease or disorder, orincreased time to progression. In other words, the therapeutic responsecan be a full or partial therapeutic response. Accordingly, atriterpene-responsive condition, disease or disorder is one againstwhich the triterpene-based composition will provide or exhibit atherapeutic response in a subject.

A therapeutic response can also be described as one in which the qualityof life of the patient afflicted with the condition, disease or disorderis improved. Improvement in quality of life may occur, for example,through a reduction in occurrence, frequency or severity of symptomsassociated with the condition, disease or disorder. For example, for aneurological condition, improvement in quality of life may be reducedtremors, reduced involuntary muscle movements, reversal of loss orpartial loss of nerve-muscle coordination, increased memory retention,etc.

The methods of the invention include methods of treating as well asprophylactic methods of preventing. Methods of treating are thosemethods wherein the subject receiving the composition of the inventionis already suffering from a specified condition, disease or disorder.Methods of preventing are those methods wherein the subject receivingthe composition of the invention is not yet suffering from a specifiedcondition, disease or disorder.

“Preventing occurrence of a condition, disease or disorder in apopulation of subjects at risk” means that said condition, disease ordisorder will not occur during a predetermined time period in ademographically predetermined population of subjects that are at risk ofsuffering from the same. The prevention during the predetermined timeperiod occurs as a result of subjects in that population having beenadministered a composition according to the invention. A composition ofthe invention can be administered prophylactically to prevent any of theconditions, diseases or disorders disclosed herein as well asetiologically-related conditions, diseases or disorders.

As one example, when a neuroprotective composition is administered for apredetermined time period to subjects in a population of subjects atrisk of suffering from stroke, stroke will not occur in those subjectsduring the predetermined time period. In particular, a neuroprotectivecomposition is chronically administered over a period of one year to apopulation of subjects at risk of suffering from Alzheimer's disease orany of the tauopathology related diseases, and the subjects in thatpopulation do not exhibit symptoms associated with Alzheimer's duringthat one-year period.

“Reducing the incidence of occurrence of a condition, disease ordisorder in a population of subjects at risk” is related in meaning to“preventing the incidence”, except that “reducing the incidence ofoccurrence” permits the occurrence of the condition, disease or disorderin a demographically predetermined population of subjects but at a rateof occurrence or a level of severity that is reduced as compared to anotherwise demographically similar predetermined population of subjectsat risk not being administered the composition according to theinvention.

As used herein, “time to progression” is the period, length or durationof time after a disease is diagnosed (or treated) until the diseasebegins to worsen. It is the period of time during which the level of adisease is maintained without further progression of the disease, andthe period of time ends when the disease begins to progress again.Progression of a disease is determined by “staging” a subject sufferingfrom a condition prior to or at initiation of therapy.

For example, the subject's neurological health is determined prior to orat initiation of therapy. The subject is then treated with theneuroprotective composition, and the neurological health monitoredperiodically. At some later point in time, the symptoms of theneurological condition may worsen, thus marking progression of thedisease and the end of the “time to progression”. The period of timeduring which the disease did not progress or during which the level orseverity of the disease did not worsen is the “time to progression”.

A dosing regimen includes a therapeutically relevant dose (ortherapeutically effective dose) of triterpene-based compositionadministered according to a dosing schedule. A therapeutically relevantdose, therefore, is a therapeutic dose at which a therapeutic responseof the condition, disease or disorder to treatment with said compositionis observed and at which a subject can be administered said compositionwithout an excessive amount of unwanted or deleterious side effects. Atherapeutically relevant dose is non-lethal to a subject, even though itmay cause some side effects (adverse events) in the patient. It is adose at which the level of clinical benefit to a subject beingadministered said composition exceeds the level of deleterious sideeffects experienced by the subject due to administration of saidcomposition.

A therapeutically relevant dose will vary from subject to subjectaccording to a variety of established pharmacologic, pharmacodynamic andpharmacokinetic principles. However, a therapeutically relevant dosewill typically be in the range of 0.1 to 100 micrograms of saidcomposition, being in either solid, liquid or semisolid form. It isknown in the art that the actual amount of a pharmacologically activecomponent/agent required to provide a target therapeutic result in asubject may vary from subject to subject according to the basicprinciples of pharmacy.

A therapeutically relevant (effective) dose can be administeredaccording to any dosing regimen typically used in the treatment ofconditions, diseases or disorders disclosed herein. A therapeuticallyrelevant dose can be administered once, twice, thrice or more dailydosing schedule. It can be administered every other day, every thirdday, every fourth day, every fifth day, semiweekly, weekly, biweekly,every three weeks, every four weeks, monthly, bimonthly, semimonthly,every three months, every four months, semiannually, annually, oraccording to a combination of any of the above to arrive at a suitabledosing schedule. For example, a therapeutically relevant dose can beadministered once daily for one or more weeks.

The examples below include evidence of the efficacy of theneuroprotective composition in treating neurological conditions such asneurological diseases, neurological disorders and stroke. Example 7details a method of treating Alzheimer's disease with a neuroprotectivecomposition or a combination of neuroprotective composition with one ormore other therapeutic agents. Example 8 details a method of treatingHuntington's disease with a neuroprotective composition or a combinationof neuroprotective composition with one or more other therapeuticagents. Example 9 details a method of treating stroke-mediated andnon-stroke mediated ischemic brain injury with a neuroprotectivecomposition or a combination of neuroprotective composition with one ormore other therapeutic agents. Example 10 details a method ofParkinson's disease with a neuroprotective composition or a combinationof neuroprotective composition with one or more other therapeuticagents.

In general, a subject having a neurological condition is treated asfollows. A subject presenting with a neurological condition is evaluatedto determine whether or not the neurological condition is Alzheimer'sdisease, Huntington's disease, stroke, Parkinson's disease or otherneurological condition. If the subject has a positive diagnosis,administration of the neuroprotective composition is indicated. Initialdoses of the composition are administered to the subject according to aprescribed dosing regimen for a period of time. The subject's clinicalresponse and level of therapeutic response are determined periodically.If the level of therapeutic response is too low at one dose, then thedose is escalated according to a predetermine dose escalation scheduleuntil the desired level of therapeutic response in the subject isachieved. If the subject exhibits undesirable side effects or anunacceptable level of side effects, then the dose is deescalated untilthe desired balance of level of therapeutic response versus side effectprofile in the subject is achieved. Treatment of the subject with theneuroprotective composition is continued as needed. The dose or dosingregimen can be adjusted as needed until the patient reaches the desiredclinical endpoint(s) such as cessation of the disease itself, reductionin disease associated symptoms, and/or a reduction in the progression ofthe disease process.

Example 3 provides a detailed description of an in vitro assay used toevaluate the efficacy of the neuroprotective composition for thetreatment of stroke-mediated ischemic neuronal injury. The assay is abrain slice-based assay for oxygen and glucose deprivation (OGD) used toinduce ≥50% loss of healthy cortical neurons by 24 hours. The samplevehicle is used as a positive control.

Various compositions were tested in OGD treated brain slices (strokemodel) and non-OGD treated (i.e. control) brain slices (non-strokemodel). Data (FIGS. 9A-9C) were obtained for Composition I (triterpenemolar ratio similar to PBI-04711), Composition II (triterpene molarratio similar to PBI-05204) and Composition III (triterpene molar ratiosimilar to PBI-01011). The data indicate that each of the compositionsprovide neuroprotection, but the improved neuroprotective composition(Composition III) provides neuroprotection across a wider dosing range(wider concentration range). For example, Composition I providesneuroprotection at 10 μM but does not provide neuroprotection at aconcentration of 1 μM or less. Composition II provides neuroprotectionat 1 μM and 10 μM but does not provide neuroprotection at aconcentration of 0.1 μM or less. On the other hand, Composition IIIprovides neuroprotection at 0.1 μM (100 Nm), 1 μM and 10 μM.

Accordingly, the improved neuroprotective composition provides a widerdosing range or wider dose response than other triterpene-basedcompositions on a total equimolar basis. The improved composition allowsfor administration of higher doses of the combination of triterpeneswithout substantially increasing the undesired adverse events (sideeffects) that might be caused by the individual triterpenes. On apractical basis, a clinician can administer high or low doses of thetriterpene mixture and still expect low occurrence of triterpene-relatedadverse events.

The invention provides a method of protecting neurons against loss ofactivity caused by oxygen depletion or oxygen-glucose depletion byexposing the oxygen depleted and/or glucose-depleted neurons to aneffective amount of improved triterpene-based neuroprotectivecomposition to minimize loss of activity, reduce the rate of loss ofactivity, stop the loss of activity, slow down onset of loss ofactivity, and/or protect the function of neurons caused by exposing theoxygen depleted and/or glucose-depleted conditions.

We determined whether the reduced neuroprotective activity ofCompositions I and II might be due to cellular toxicity of one or moreof the individual triterpenes. We conducted a direct study of thecellular toxicities of oleanolic acid (OA), ursolic acid (UA), andbetulinic acid (BA) using two independent measures of cell death:propidium iodide (PI) staining for nuclear breakdown and the MTS assayfor cell metabolic activity. PI enters cells with damaged membranes andstains DNA, thereby detecting dead/dying cells using image-basedhigh-content assays on the Cellomics Arrayscan VTI. The MTS assay is awell-based assay in which cleavage of a tetrazolium reporter bymitochondrial enzymes produces colorimetric readout and reports relativenumbers of healthy cells. The data (FIGS. 6A and 6B for UA, FIGS. 7A and7B for BA; FIGS. 8A and 8B for OA) confirmed that UA and BA can behighly toxic compounds on a dose dependent manner, each leading to 50%loss of metabolic activity in the 5-15 M range, whereas no significantreduction in MTS activity was observed for OA through 75 M, the highestconcentration of OA tested.

The potential additive and synergistic performance of the mixture oftriterpenes was determined by analyzing their performance individuallyand in different mixtures by way of a gene expression assay. Wedetermined the relative activity of the various compositions to induceARE gene expression of Hmox1 and Srxn at various differentconcentrations (μM). Activation of the ARE genes by ursolic acid andbetulinic acid was examined using more closely-spaced concentrationsteps. Betulinic acid, like ursolic acid, is able to induce clearupregulation of Srx and Hmox1 despite its toxicity at higherconcentrations (FIG. 4). The data (FIGS. 10A and 10B) indicated that OAas a single agent was not able to induce appreciable ARE gene expressionat any concentration tested. Even at low concentration, UA and BA causedcellular toxicity. A number of combinations of OA+UA and OA+UA+BA didinduce significant degrees of ARE gene expression, but did so to extentswell >10-fold by the highest concentration tested for that condition(striped, red bars for Srxn1). Such excessive induction is associatedwith longer term cellular toxicity by 24 hours of treatment. Thisincluded UA tested as a single agent at 9 M. Of the mixtures tested,only 10:1:1 OA:UA:BA induced significant induction of ARE genes but toan extent <5-10 fold within the full concentration range tested Thus,the 10:1:1 OA:UA:BA mixture satisfied both criteria for the targetprofile proposed, namely: 1) that for this ratio no single componentwould be dose-limiting due to toxicity; and 2) that ARE target genes aresignificantly induced but by not more than 5- to 10-fold at the highest.Other closely related molar ratios within the ranges described hereinalso provide substantial neuroprotection without excessive cellulartoxicity. Significant induction of the ARE-luciferase reporter is seenin similar concentration ranges that provided neuroprotection in theOGD, APP, and tau brain slice neuroprotection assays.

The results herein were surprising and unexpected. We thus determinedthat the improved Composition III and other closely related compositions(those having a higher molar content of oleanolic acid and asubstantially lower molar content of ursolic acid and betulinic acid)simultaneously provide comparatively reduced cellular toxicity at higherdose and comparatively increased efficacy at very low concentration.This means that on a total-triterpene equimolar basis, the improvedcomposition of the invention provides substantially improved clinicalbenefit over the individual triterpenes and even over triterpene-basedcompositions having triterpene molar ratios outside the ranges describedherein.

Efficacy of the improved composition of the invention in treating arange of conditions, diseases or disorders can be established byemploying known respective prognostic assays (in vitro, in vivo or exvivo), performing animal studies, and/or performing clinical studies inhumans. Examples 16-34 provide exemplary literature methods suitable forestablishing the therapeutic efficacy provided by the improvedcomposition of the invention.

During each such evaluation, the individual triterpenes (oleanolic acid,ursolic acid and betulinic acid) are used as control samples toestablish baseline activity thereof. Combinations of two or more of thetriterpenes are then evaluated. Generalized (molar ratios unspecified)exemplary sample compositions for each assay are set forth in the tablebelow.

Triterpene Present (Y/N) Oleanolic Ursolic Betulinic Sample acid (O)acid (U) acid (B) Control 1 Y N N Control 2 N Y N Control 3 N N Y 1 Y YN 2 Y N Y 3 Y Y Y 4 N Y Y

Examples 1 and 2 provide detailed exemplary compositions with the molarratio(s) of the triterpenes as specified therein.

In some embodiments, the composition of the invention comprises at leasttwo triterpenes (free acid, salt(s), derivative(s), and/or prodrug(s)thereof). For example, the invention provides a composition comprising acombination of at least ursolic acid and oleanolic acid or a combinationof at least betulinic acid and oleanolic acid, wherein oleanolic acid ispresent in substantial molar excess over ursolic acid and betulinicacid, respectively.

In some embodiments, the composition of the invention comprises at leastthree triterpenes (free acid, salt(s), derivative(s), and/or prodrug(s)thereof). In some embodiments, the neuroprotective composition of theinvention comprises oleanolic acid, ursolic acid and at least one othertriterpene. For example, the composition can further comprise betulinicacid or at least one other triterpene.

The composition can exclude cardiac glycoside, pharmacologically activepolysaccharide, and steroid. For example, the neuroprotectivecomposition excludes oleandrin, neriifolin or pharmacologically activepolysaccharide obtained from Nerium species plant.

When oleanolic acid (O; free acid, salt, derivative, and/or prodrugthereof) and ursolic acid (U; free acid, salt, derivative, and/orprodrug thereof) are present as the primary or sole triterpenes in thecomposition, the molar ratio of O:U can be varied and can range fromabout 9-12:about 0.33-5, about 9-12:about 0.4-5, about 9-12:about 0.5-5,about 9-12:about 0.7-5, about 9-12:about 0.9-5, about 9-12:about 1-5,about 9-12:about 0.33-4, about 9-12:about 0.4-4, about 9-12:about 0.5-4,about 9-12:about 0.7-4, about 9-12:about 0.9-4, about 9-12:about 1-4,about 9-12:about 0.33-3, about 9-12:about 0.4-3, about 9-12:about 0.5-3,about 9-12:about 0.7-3, about 9-12:about 0.9-3, about 9-12:about 1-3,about 9-12:about 0.33-2, about 9-12:about 0.4-2, about 9-12:about 0.5-2,about 9-12:about 0.7-2, about 9-12:about 0.9-2, about 9-12:about 1-2,about 10:about 1-5, about 10:about 1-3, about 9-12:about 0.5-1.5, about9-11:about 0.5-1.5, about 9.5-10.5:about 0.75-1.25, about 9.5-10.5:about0.8-1.2, or about 9.75-10.5:about 0.9-1.1.

When oleanolic acid (O; free acid, salt, derivative, and/or prodrugthereof) and betulinic acid (B; free acid, salt, derivative, and/orprodrug thereof) are present as the primary or sole triterpenes in thecomposition, the molar ratio of O:B can be varied and can range fromabout 9-12:about 0.33-5, about 9-12:about 0.4-5, about 9-12:about 0.5-5,about 9-12:about 0.7-5, about 9-12:about 0.9-5, about 9-12:about 1-5,about 9-12:about 0.33-4, about 9-12:about 0.4-4, about 9-12:about 0.5-4,about 9-12:about 0.7-4, about 9-12:about 0.9-4, about 9-12:about 1-4,about 9-12:about 0.33-3, about 9-12:about 0.4-3, about 9-12:about 0.5-3,about 9-12:about 0.7-3, about 9-12:about 0.9-3, about 9-12:about 1-3,about 9-12:about 0.33-2, about 9-12:about 0.4-2, about 9-12:about 0.5-2,about 9-12:about 0.7-2, about 9-12:about 0.9-2, about 9-12:about 1-2,about 10:about 1-5, about 10:about 1-3, about 9-12:about 0.5-1.5, about9-11:about 0.5-1.5, about 9.5-10.5:about 0.75-1.25, about 9.5-10.5:about0.8-1.2, or about 9.75-10.5:about 0.9-1.1.

When oleanolic acid (O; free acid, salt, derivative, and/or prodrugthereof), ursolic acid (U; free acid, salt, derivative, and/or prodrugthereof) and betulinic acid (B; free acid, salt, derivative, and/orprodrug thereof) are present as the primary or sole triterpenes in thecomposition, the molar ratio oleanolic acid:ursolic acid:betulinic acidis about 10:about 1:about 1, about 9-11:about 0.5-1.5:about 0.5-1.5,about 9.5-10.5:about 0.75-1.25:about 0.75-1.25, about 9.5-10.5:about0.8-1.2:about 0.8-1.2, about 9.75-10.5:about 0.9-1.1:about 0.9-1.1,about 9-12:about 0.15-2.5:about 0.15-2.5, about 9-12:about 0.2-2.5:about0.2-2.5, about 9-12:about 0.25-2.5:about 0.25-2.5, about 9-12:about0.35-2.5:about 0.35-2.5, about 9-12:about 0.45-2.5:about 0.45-2.5, about9-12:about 0.5-5:about 0.5-2.5, about 9-12:about 0.16-2:about 0.16-2,about 9-12:about 0.2-2:about 0.2-2, about 9-12:about 0.25-2:about0.25-2, about 9-12:about 0.25-2:about 0.25-2, about 9-12:about0.45-2:about 0.45-2, about 9-12:about 0.5-2:about 0.5-2, about9-12:about 0.16-1.5:about 0.16-1.5, about 9-12:about 0.2-1.5:about0.2-1.5, about 9-12:about 0.25-1.5:about 0.25-1.5, about 9-12:about0.7-1.5:about 0.35-1.5, about 9-12:about 0.45-1.5:about 0.45-1.5, about9-12:about 0.5-1.5:about 0.5-1.5, about 9-12:about 0.16-1:about 0.16-1,about 9-12:about 0.2-1:about 0.2-1, about 9-12:about 0.25-1:about0.25-1, about 9-12:about 0.35-1:about 0.35-1, about 9-12:about0.45-1:about 0.45-1, about 9-12:about 0.5-1:about 0.5-1, about 10:about0.5-2.5:about 0.5-2.5, about 10:about 0.1-1.5:about 0.1-1.5, about9-12:about 0.25-0.75:about 0.25-0.75, about 9.5-10.5:about0.35-0.7:about 0.35-0.7, about 9.5-10.5:about 0.4-0.6:about 0.4-0.6, orabout 9.75-10.5:about 0.45-0.6:about 0.45-0.6.

Example 11 provides a detailed description of an in vitro assay used toevaluate the efficacy of the neuroprotective composition for thetreatment of Alzheimer's disease. The assay is a brain slice-based assayfor APP/Aβ-induced (APP: amyloid precursor protein) degeneration ofcortical pyramidal neurons. Upon cleavage by a secretase enzyme, the APPis reduced to Aβ peptides which are believed to be a causative factor inbeta-amyloid plaque formation. Aβ proteins are associated withbeta-amyloid plaque formation and are believed to be a hallmark if notetiologic factor in Alzheimer's disease. Biolistic transfection is usedto introduce vital markers such as YFP (a marker yellow fluorescentprotein) and to introduce disease gene constructs into the same neuronalpopulations in the brain slices. YFP is co-transfected with APP isoformsleading to the progressive degeneration of cortical pyramidal neuronsover the course of three to four days after brain slice preparation andtransfection. The data indicate that the neuroprotective compositionprovides a concentration-dependent neuroprotection to APP-transfectedbrain slices. Composition III (O:U:B molar ratio of about 10:1:1 as inPBI-01011) provides greater neuroprotection than Composition II (O:U:Bmolar ratio of about 7.8:7.4:1 as in PBI-05204) and greaterneuroprotection than Composition I (O:U:B molar ratio of about 3:2.2:1as in PBI-04711). The data are of significance in that few compounds ortherapeutic strategies in the literature have shown any significantprotection of neurons in this in vitro assay representative of Alzheimerdisease.

The composition of the invention also provides strong neuroprotection intwo additional brain slice models in which cortical neuronaldegeneration is driven by biolistic transfection of expressionconstructs for genes implicated in CNS neurodegeneration, namely,amyloid precursor protein (APP) and tau. In these models, APP and tautransfection induces progressive neurodegeneration of cortical neuronsover the course of 3-4 days, in contrast to the neuronal injury anddeath caused by OGD which occurs over a 24 h period in the brain slicemodel.

Data indicate that the composition provides neuroprotection in thisassay, even though it does not contain any cardiac glycosides. Theneuroprotective composition provides significant concentration-dependentneuroprotection in both the APP and tau brain slice neurodegenerationmodels.

The composition is evaluated with the tau4R brain slice-basedAlzheimer's assay similar to the APP assay except that the Tau constructis used (Example 11). The number of healthy cortical neurons isdetermined. Efficacy in this assay is defined as or based upon therelative total number of healthy versus unhealthy number and percentageof degraded neurons in the presence of varying amounts ofneuroprotective composition. The negative control in these experimentsconsisted of brain slices that were not exposed to OGD while brainslices exposed to OGD but not treated with neuroprotective compositionserved as the internal positive control. The neuroprotective compositionprovides neuroprotection in this assay.

Accordingly, the invention provides a method of protecting neuronsagainst loss of activity caused by Alzheimer's disease, the methodcomprising: exposing the neurons exhibiting characteristics ofAlzheimer's disease to an effective amount of triterpene-basedneuroprotective composition to minimize loss of activity, reduce therate of loss of activity, stop the loss of activity, slow down onset ofloss of activity, and/or critical functioning of the neurons caused byAlzheimer's disease. In some embodiments, the method employs aneffective amount of neuroprotective composition.

Example 6 provides a detailed description of an assay used to evaluatethe efficacy of the neuroprotective composition for the treatment ofHuntington's disease. Mutant htt protein is introduced viaelectroporation into high-density, mixed co-cultures of corticalneurons, striatal neurons, and glia. The striatal and cortical neuronsare transfected with different color fluorescent proteins therebyfacilitating the separate identification of the different types ofneurons in the co-culture. The color fluorescent proteins arefluorescent and ‘emit’ color upon activation with a light source ofappropriate wavelength. The data indicate that the neuroprotectivecomposition can be used to treat Huntington's disease.

Accordingly, the invention provides a method of protecting neuronsagainst loss of activity caused by Huntington's disease, the methodcomprising: exposing the neurons exhibiting characteristics ofHuntington's disease to an effective amount of neuroprotectivecomposition to minimize loss of activity, reduce the rate of loss ofactivity, stop the loss of activity, slow down onset of loss ofactivity, and/or normal function of the neurons caused by Huntington'sdisease.

Examples 3, 4 and 12 detail an exemplary brain-slice assay that can beused to evaluate the efficacy of neuroprotective composition in thetreatment of stroke in a subject following completion of a delay periodafter the stroke. The brain-slice assay with oxygen glucose deprivationis conducted as described herein; however, rather than treating thebrain slices prophylactically with the composition, they are treatedwith the composition after delay periods of 0, 1, 2, 4, and 6 hours. Thedata should demonstrate that the neuroprotective composition iseffective at providing significant neuroprotection for delay periods ofup to 1, up to 2, up to 3, up to 4, up to 5, up to about 6 hours afterthe stroke.

Accordingly, the invention provides a time-delayed method of treatingstroke in a subject by administration of a dose of neuroprotectivecomposition to a subject after the subject has suffered a stroke. Withinan acceptable delay period after a subject has suffered the stroke, aninitial dose of the neuroprotective composition is administeredaccording to an initial dosing regimen. Then, adequacy of the subject'sclinical response and/or therapeutic response to treatment with thecomposition is determined. If the subject's clinical response and/ortherapeutic response is adequate, then treatment with the composition iscontinued as needed until the desired clinical endpoint is achieved.Alternatively, if the subject's clinical response and/or therapeuticresponse are inadequate at the initial dose and initial dosing regimen,the dose is escalated or deescalated until the desired clinical responseand/or therapeutic response in the subject is achieved. Dose escalationor de-escalation can be performed in conjunction with a change in thedosing regimen, such as a change in dosing frequency or overall periodof dose administration.

Some of the brain slice assays herein are conducted under conditionswherein the brain tissue is treated with the neuroprotective compositionprior to OGD. Under those conditions, the data establishes the utilityof the neuroprotective composition at prophylactically providingneuroprotection against damage caused by stroke.

The inventors have discovered that the composition of the inventionprovides neuroprotection mediated through antioxidant transcriptionalresponse elements (AREs) with triterpene(s). The triterpene(s) alsoinduces nuclear factor erythroid 2 related factor 2 (Nrf2)-dependentantioxidant genes to provide neuroprotection. When the neuroprotectivecomposition is administered to a subject in need thereof, thecomposition provides neuroprotection via at least a two-fold mechanism.When the neuroprotective composition is administered to a subject inneed thereof, the composition provides neuroprotection at least throughARE up-regulation.

If a clinician intends to treat a subject having a neurologicalcondition with a combination of triterpene-based composition and one ormore other therapeutic agents, and it is known that the particularcondition, disease or disorder, which the subject has, is at leastpartially therapeutically responsive to treatment with said one or moreother therapeutic agents, then the present method invention comprises:administering to the subject in need thereof a therapeutically relevantdose of triterpene-based composition and a therapeutically relevant doseof said one or more other therapeutic agents, wherein thetriterpene-based composition is administered according to a first dosingregimen and the one or more other therapeutic agents is administeredaccording to a second dosing regimen. In some embodiments, the first andsecond dosing regimens are the same. In some embodiments, the first andsecond dosing regimens are different.

If the neurological condition being treated is Alzheimer's disease, theone or more other therapeutic agents can be selected from the groupconsisting of BACE inhibitors or acetylcholinesterase inhibitors. Insome embodiments, the one or more other therapeutic agents can beselected from the group consisting of Namenda™ (memantine HCl), Aricept™(donepezil), Razadyne™ (galantamine), Exelon™ (rivastigmine),and Cognex™(tacrine).

If the neurological condition being treated is Huntington's disease, theone or more other therapeutic agents can be selected from the groupconsisting of natural products, anticonvulsants, NMDA (n-methyld-aspartate) receptor antagonists, and sodium channel blockers.Exemplary agents include Vitamin E, Baclofen (a derivative of CoQ10),Lamotrigine (an anticonvulsant), remacemide (an anesthetic which is alow affinity NMDA antagonist), and riluzole (Na channel blocker). Theefficacy of each of these agents is considered to be low (Mestre T. etal, Chochrane Database Systematic Reviews Jul. 8, 2009; 8(3): CD006455)on its own; however, it is expected that administration of a dosage formcontaining neuroprotective composition to subjects receiving one or moreof these other agents will provide a subject, having a neurologicaldisorder, an improved clinical affect as compared to administration ofthese agents absent the neuroprotective composition.

If the neurological condition being treated is stroke-mediated ischemicbrain injury (ischemic stroke), then the therapeutic treatmentsdisclosed in the literature (Gutierrez M. et al. “Cerebral protection,brain repair, plasticity and cell therapy in ischemic stroke”Cerebrovasc. Dis. 2009; 27 Suppl 1:177-186), e.g. intravenousthrombolysis, can be employed in addition to the neuroprotectivecomposition. In some embodiments, the one or more other therapeuticagents can be selected from the group consisting of drugs such asAlteplase (a thrombolytic agent).

If the neurological condition being treated is Parkinson's disease, theone or more other therapeutic agents include a combination of carbidopaand levodopa, rasagiline, pramipexole, ropinrole, amantadine, memantine,entacapone, rotigotine, benztropine, selegiline, biperiden, acombination of carbidopa and levodopa and entacapone, trihexylphenidyl,rivastigmine, apomorphine, levodopa, carbidopa, bromocriptine,belladonna, tolcapone, or a combination thereof.

The one or more other therapeutic agents can be selected from the groupconsisting of BACE (beta-secretase 1; beta-site amyloid precursorprotein cleaving enzyme 1, beta-site APP cleaving enzyme 1,membrane-associated aspartic protease 2, memapsin-2, aspartyl protease2, and ASP2) inhibitors, AZD3293, acetylcholinesterase inhibitors,Namenda™ (memantine HCl), Aricept™ (donepezil), Razadyne™ (galantamine),Exelon™ (rivastigmine), Cognex™ (tacrine), anticonvulsants, NMDA(n-methyl d-aspartate) receptor antagonists, sodium channel blockersVitamin E, Baclofen (a derivative of CoQ10), Lamotrigine (ananticonvulsant), remacemide (an anesthetic which is a low affinity NMDAantagonist), riluzole (Na channel blocker), Alteplase (a thrombolyticagent), levodopa, carbidopa, amantadine, COMT (catechol O-methyltransferase) inhibitor, tolcapone, entacapone, opicapone, dopamineagonist, bromocriptine, pergolide, pramipexole, ropinirole, piribedil,cabergoline, apomorphine, lisuride, MAO-B (monoamine oxidase-B)inhibitor (selective and non-selective MAO-B inhibitors),anticholinergic, cholinesterase inhibitor, isocarboxazid, nialamide,phenelzine, hydracarbazine, rasagiline, selegiline, linezolid, or acombination thereof.

Compounds or combinations of compounds suitable for cotherapy oradjunctive use with the triterpene-based composition are disclosed byDrugs.com (https://www.drugs.com/drug-classes.html) and are readilyidentifiable by conducting drug class searches or specific compoundsearches. For example, a search for suitable antiviral compounds in theDrugs.com database resulted in identification of adamantane-basedantiviral agent, interferon-based antiviral agent, chemokine receptorantagonist, integrase strand transfer inhibitor, neuraminidaseinhibitor, non-nucleoside reverse transcriptase inhibitor, NS5Ainhibitor, nucleoside reverse transcriptase inhibitor, proteaseinhibitor, purine nucleoside, or a combination thereof. One or more ofthose antiviral compounds can be coadministered or used as cotherapy orused adjunctively with the triterpene-based composition.

Additionally, the one or more other therapeutic agents that are approvedfor human use are listed, searchable and viewable in the databases ofthe U.S. Food and Drug Administration (U.S.F.D.A.), World HealthOrganization (W.H.O.), European Medicines Agency (E.M.E.A.), TherapeuticGoods Administration (TGA, Australia), Pan American Health Organization(PAHO), Medicines and Medical Devices Safety Authority (Medsafe, NewZealand) or the various Ministries of Health worldwide, the entiredisclosures of which are hereby incorporated by reference.

The one or more other therapeutic agents can be administered at dosesand according to dosing regimens that are clinician-recognized as beingtherapeutically effective or at doses that are clinician-recognized asbeing sub-therapeutically effective. The clinical benefit and/ortherapeutic effect provided by administration of a combination of thetriterpene-based composition and one or more other therapeutic can beadditive or synergistic, such level of benefit or effect beingdetermined by comparison of administration of the combination toadministration of the individual triterpene-based composition and one ormore other therapeutic agents. The one or more other therapeutic agentscan be administered at doses and according to dosing regimens assuggested or described by the U.S. Food and Drug Administration(U.S.F.D.A.), World Health Organization (W.H.O.), European MedicinesAgency (E.M.E.A.), Therapeutic Goods Administration (TGA, Australia),Pan American Health Organization (PAHO), Medicines and Medical DevicesSafety Authority (Medsafe, New Zealand) or the various Ministries ofHealth worldwide.

A triterpene-based composition of the invention can be prepared bymixing the individual components thereof into a mixture (Examples 1-2).For example, the triterpene-based composition can be prepared by mixingat least oleanolic acid and ursolic acid, and optionally betulinic acid,according to the molar ratios described herein. Alternatively, thetriterpene-based composition can be prepared by mixing at leastoleanolic acid and betulinic acid, and optionally ursolic acid,according to the molar ratios described herein. The triterpene-basedcomposition can also be prepared by mixing at least oleanolic acid,ursolic acid, and betulinic acid, according to the molar ratiosdescribed herein.

We have determined that triterpenes exhibit different levels of activityin the neuroprotection OGD assay described herein. Accordingly, thelevel of contribution of the individual triterpenes toward efficacy andcellular toxicity of a neuroprotective composition containing them. Wehave discovered that the molar ratio of the triterpenes in thetriterpene-based neuroprotective composition must be correctly balancedin order to provide the greatest level of neuroprotective efficacy whilemaintaining reduced level of cellular toxicity.

The lower activity of UA in the neuroprotection OGD assay is surprising.Data obtained from the expression assays demonstrate that Fraction 0-4induces substantial expression of Nrf2, Srx and Hmox1 and lowerexpression of Gclc and Nqo1; however, data also demonstrate that theinduction of Srx and Hmox1 is due more so to the activity of UA than ofOA or BA. The efficacy (especially the broad dose response curve andhigh level of efficacy at low concentrations) of the compositioncomprising plural triterpenes is apparently due to various mechanismsoperating synergistically to provide neuroprotection.

We have discovered that the level of efficacy provided by thecompositions of the invention can be improved by employing compositionspossessing suitable molar ratios of O:U or of O:U:B or of O:B. Solutionscontaining the following molar ratios of triterpene(s) were prepared(Examples 1-2) and evaluated for neuroprotective activity and ARE geneinduction activity as described herein.

In some embodiments, an improved triterpene-based composition comprisesat least oleanolic acid (free acid, salt(s) thereof, derivative(s)thereof, and/or prodrug(s) thereof) and ursolic acid (free acid, salt(s)thereof, derivative(s) thereof, and/or prodrug(s) thereof) present at amolar ratio of OA to UA as described herein, wherein OA is present inlarge molar excess over UA.

In some embodiments, an improved triterpene-based composition comprisesat least oleanolic acid (free acid, salt(s) thereof, derivative(s)thereof, and/or prodrug(s) thereof) and betulinic acid (free acid,salt(s) thereof, derivative(s) thereof, and/or prodrug(s) thereof)present at a molar ratio of OA to BA as described herein, wherein OA ispresent in large molar excess over BA.

In some embodiments, an improved triterpene-based composition comprisesat least oleanolic acid (free acid, salt(s) thereof, derivative(s)thereof, and/or prodrug(s) thereof), ursolic acid (free acid, salt(s)thereof, derivative(s) thereof, and/or prodrug(s) thereof), andbetulinic acid (free acid, salt(s) thereof, derivative(s) thereof,and/or prodrug(s) thereof) present at a molar ratio of OA to UA to BA asdescribed herein, wherein OA is present in large molar excess over bothUA and BA.

An improved triterpene-based composition provides a wider dose responsecurve than the respective triterpenes individually and provides reducedcellular toxicity when compared on a total equimolar basis, especiallywhen compared at the higher end of the dosing range.

The triterpene-based composition can be formulated in any suitablepharmaceutically acceptable dosage form. Parenteral, otic, ophthalmic,nasal, inhalable, buccal, sublingual, enteral, topical, oral, peroral,and injectable dosage forms are particularly useful. Particular dosageforms include a solid or liquid dosage forms. Exemplary suitable dosageforms include tablet, capsule, osmotic device, pill, caplet, troche,sache, solution, suspension, dispersion, vial, bag, bottle, injectableliquid, i.v. (intravenous), i.m. (intramuscular) or i.p.(intraperitoneal) administrable liquid and other such dosage forms knownto the artisan of ordinary skill in the pharmaceutical sciences.

Suitable dosage forms containing the triterpene-based composition can beprepared by mixing the said composition with pharmaceutically acceptableexcipients as described herein or as described in Pi et al. (“Ursolicacid nanocrystals for dissolution rate and bioavailability enhancement:influence of different particle size” in Curr. Drug Deliv. (March 2016),13(8), 1358-1366), Yang et al. (“Self-microemulsifying drug deliverysystem for improved oral bioavailability of oleanolic acid: design andevaluation” in Int. J. Nanomed. (2013), 8(1), 2917-2926), Li et al.(Development and evaluation of optimized sucrose ester stabilizedoleanolic acid nanosuspensions prepared by wet ball milling with designof experiments” in Biol. Pharm. Bull. (2014), 37(6), 926-937), Zhang etal. (“Enhancement of oral bioavailability of triterpene through lipidnanospheres: preparation, characterization, and absorption evaluation”in J. Pharm. Sci. (June 2014), 103(6), 1711-1719), Godugu et al.(“Approaches to improve the oral bioavailability and effects of novelanticancer drugs berberine and betulinic acid” in PLoS One (March 2014),9(3):e89919), Zhao et al. (“Preparation and characterization of betulinnanoparticles for oral hypoglycemic drug by antisolvent precipitation”in Drug Deliv. (September 2014), 21(6), 467-479), Yang et al.(“Physicochemical properties and oral bioavailability of ursolic acidnanoparticles using supercritical anti-solvent (SAS) process” in FoodChem. (May 2012), 132(1), 319-325), Cao et al. (“Ethylene glycol-linkedamino acid diester prodrugs of oleanolic acid for PEPT1-mediatedtransport: synthesis, intestinal permeability and pharmacokinetics” inMol. Pharm. (August 2012), 9(8), 2127-2135), Li et al. (“Formulation,biological and pharmacokinetic studies of sucrose ester-stabilizednanosuspensions of oleanolic acid” in Pharm. Res. (August 2011), 28(8),2020-2033), Tong et al. (“Spray freeze drying with polyvinylpyrrolidoneand sodium caprate for improved dissolution and oral bioavailablity ofoleanolic acid, a BCS Class IV compound” in Int. J. Pharm. (February2011), 404(1-2), 148-158), Xi et al. (Formulation development andbioavailability evaluation of a self-nanoemulsified drug delivery systemof oleanolic acid” in AAPS PharmSciTech (2009), 10(1), 172-182), Chen etal. (“Oleanolic acid nanosuspensions: preparation, in-vitrocharacterization and enhanced hepatoprotective effect” in J. Pharm.Pharmacol. (February 2005), 57(2), 259-264), the entire disclosures ofwhich are hereby incorporated by reference.

Suitable dosage forms can also be made according to U.S. Pat. No.8,187,644 B2 to Addington, which issued May 29, 2012, U.S. Pat. No.7,402,325 B2 to Addington, which issued Jul. 22, 2008, U.S. Pat. No.8,394,434 B2 to Addington et al, which issued Mar. 12, 2013, the entiredisclosures of which are hereby incorporated by reference. Suitabledosage forms can also be made as described in Examples 13-15.

The desired dose for oral administration is up to 5 dosage formsalthough as few as one and as many as ten dosage forms may beadministered as a single dose. Exemplary dosage forms contain 0.01-5 or0.01-10 mg of the triterpene-based composition per dosage form, for atotal 0.1 to 500 mg (1 to 10 dose levels) per dose. Doses will beadministered according to dosing regimens that may be predeterminedand/or tailored to achieve specific therapeutic response or clinicalbenefit in a subject.

Some embodiments of the dosage form are not enteric coated and releasetheir charge of triterpene-based composition within a period of 0.5 to 1hours or less. Some embodiments of the dosage form are enteric coatedand release their charge of triterpene-based composition downstream ofthe stomach, such as from the jejunum, ileum, small intestine, and/orlarge intestine (colon). Enterically coated dosage forms will releasesaid composition into the systemic circulation within 1-10 hr after oraladministration.

The triterpene-based composition can be included in a rapid release,immediate release, controlled release, sustained release, prolongedrelease, extended release, burst release, continuous release, slowrelease, or pulsed release dosage form or in a dosage form that exhibitstwo or more of those types of release. The release profile of triterpenefrom the dosage form can be a zero order, pseudo-zero, first order,pseudo-first order or sigmoidal release profile. The plasmaconcentration profile for triterpene in a subject to which theneuroprotective composition is administered can exhibit one or moremaxima.

The amount of triterpene-based composition incorporated in a dose willbe in at least one or more dosage forms and can be selected according toknown principles of pharmacy. An effective amount or therapeuticallyrelevant amount of therapeutic compound is specifically contemplated. Bythe term “effective amount”, it is understood that, with respect to, forexample, pharmaceuticals, a pharmaceutically effective amount iscontemplated. A pharmaceutically effective amount is the amount orquantity of active ingredient which is enough for the required ordesired therapeutic response, or in other words, the amount, which issufficient to elicit an appreciable biological response when,administered to a patient. The appreciable biological response may occuras a result of administration of single or multiple doses of an activesubstance. A dose may comprise one or more dosage forms. It will beunderstood that the specific dose level for any patient will depend upona variety of factors including the indication being treated, severity ofthe indication, patient health, age, gender, weight, diet,pharmacological response, the specific dosage form employed, and othersuch factors.

The triterpene-based composition can be administered at low to high dosedue to the improved combination of triterpenes present and the molarratio at which they are present. A therapeutically effective dose forhumans is approximately 100-1000 mg of triterpene-based composition perKg of body weight. Such a dose can be administered up to 10 times in a24-hour period.

It should be noted that a compound herein might possess one or morefunctions in the formulation of the invention. For example, a compoundmight serve as both a surfactant and a water miscible solvent or as botha surfactant and a water immiscible solvent.

A liquid composition can comprise one or more pharmaceuticallyacceptable liquid carriers. The liquid carrier can be an aqueous,non-aqueous, polar, non-polar, and/or organic carrier. Liquid carriersinclude, by way of example and without limitation, a water misciblesolvent, water immiscible solvent, water, buffer and mixtures thereof.

As used herein, the terms “water soluble solvent” or “water misciblesolvent”, which terms are used interchangeably, refer to an organicliquid which does not form a biphasic mixture with water or issufficiently soluble in water to provide an aqueous solvent mixturecontaining at least five percent of solvent without separation of liquidphases. The solvent is suitable for administration to humans or animals.Exemplary water soluble solvents include, by way of example and withoutlimitation, PEG (poly(ethylene glycol)), PEG 400 (poly(ethylene glycolhaving an approximate molecular weight of about 400), ethanol, acetone,alkanol, alcohol, ether, propylene glycol, glycerin, triacetin,poly(propylene glycol), PVP (poly(vinyl pyrrolidone)),dimethylsulfoxide, N,N-dimethylformamide, formamide,N,N-dimethylacetamide, pyridine, propanol, N-methylacetamide, butanol,soluphor (2-pyrrolidone), pharmasolve (N-methyl-2-pyrrolidone).

As used herein, the terms “water insoluble solvent” or “water immisciblesolvent”, which terms are used interchangeably, refer to an organicliquid which forms a biphasic mixture with water or provides a phaseseparation when the concentration of solvent in water exceeds fivepercent. The solvent is suitable for administration to humans oranimals. Exemplary water insoluble solvents include, by way of exampleand without limitation, medium/long chain triglycerides, oil, castoroil, corn oil, vitamin E, vitamin E derivative, oleic acid, fatty acid,olive oil, softisan 645 (Diglyceryl Caprylate/Caprate/Stearate/Hydroxystearate adipate), miglyol, captex (Captex 350: GlycerylTricaprylate/Caprate/Laurate triglyceride; Captex 355: GlycerylTricaprylate/Caprate triglyceride; Captex 355 EP/NF: GlycerylTricaprylate/Caprate medium chain triglyceride).

Suitable solvents are listed in the “International Conference onHarmonisation of Technical Requirements for Registration ofPharmaceuticals for Human Use (ICH) guidance for industry Q3CImpurities: Residual Solvents” (1997), which makes recommendations as towhat amounts of residual solvents are considered safe inpharmaceuticals. Exemplary solvents are listed as class 2 or class 3solvents. Class 3 solvents include, for example, acetic acid, acetone,anisole, 1-butanol, 2-butanol, butyl acetate, tert-butlymethyl ether,cumene, ethanol, ethyl ether, ethyl acetate, ethyl formate, formic acid,heptane, isobutyl acetate, isopropyl acetate, methyl acetate,methyl-1-butanol, methylethyl ketone, methylisobutyl ketone,2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, orpropyl acetate.

Other materials that can be used as water immiscible solvents in theinvention include: Captex 100: Propylene Glycol Dicaprate; Captex 200:Propylene Glycol Dicaprylate/Dicaprate; Captex 200 P: Propylene GlycolDicaprylate/Dicaprate; Propylene GlycolDicaprylocaprate; Captex 300:Glyceryl Tricaprylate/Caprate; Captex 300 EP/NF: GlycerylTricaprylate/Caprate Medium Chain Triglycerides; Captex 350: GlycerylTricaprylate/Caprate/Laurate; Captex 355: Glyceryl Tricaprylate/Caprate;Captex 355 EP/NF: Glyceryl Tricaprylate/Caprate Medium ChainTriglycerides; Captex 500: Triacetin; Captex 500 P: Triacetin(Pharmaceutical Grade); Captex 800: Propylene Glycol Di(2-Ethythexanoate); Captex 810 D: GlycerylTricaprylate/Caprate/Linoleate; Captex 1000: Glyceryl Tricaprate; CaptexCA: Medium Chain Triglycerides; Captex MCT-170: Medium ChainTriglycerides; Capmul GMO: Glyceryl Monooleate; Capmul GMO-50 EP/NF:Glyceryl Monooleate; Capmul MCM: Medium Chain Mono- & Diglycerides;Capmul MCM C8: Glyceryl Monocaprylate; Capmul MCM C10: GlycerylMonocaprate; Capmul PG-8: Propylene Glycol Monocaprylate; Capmul PG-12:Propylene Glycol Monolaurate; Caprol 10G10O: Decaglycerol Decaoleate;Caprol 3GO: Triglycerol Monooleate; Caprol ET: Polyglycerol Ester ofMixed Fatty Acids; Caprol MPGO: Hexaglycerol Dioleate; Caprol PGE 860:Decaglycerol Mono-, Dioleate.

As used herein, a “surfactant” refers to a compound that comprises polaror charged hydrophilic moieties as well as non-polar hydrophobic(lipophilic) moieties; i.e., a surfactant is amphiphilic. The termsurfactant may refer to one or a mixture of compounds. A surfactant canbe a solubilizing agent, an emulsifying agent or a dispersing agent. Asurfactant can be hydrophilic or hydrophobic.

The hydrophilic surfactant can be any hydrophilic surfactant suitablefor use in pharmaceutical compositions. Such surfactants can be anionic,cationic, zwitterionic or non-ionic, although non-ionic hydrophilicsurfactants are presently preferred. As discussed above, these non-ionichydrophilic surfactants will generally have HLB values greater thanabout 10. Mixtures of hydrophilic surfactants are also within the scopeof the invention.

Similarly, the hydrophobic surfactant can be any hydrophobic surfactantsuitable for use in pharmaceutical compositions. In general, suitablehydrophobic surfactants will have an HLB value less than about 10.Mixtures of hydrophobic surfactants are also within the scope of theinvention.

Examples of additional suitable solubilizer include: alcohols andpolyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethyleneglycol, propylene glycol, butanediols and isomers thereof, glycerol,pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide,polyethylene glycol, polypropylene glycol, polyvinylalcohol,hydroxypropyl methylcellulose and other cellulose derivatives,cyclodextrins and cyclodextrin derivatives; ethers of polyethyleneglycols having an average molecular weight of about 200 to about 6000,such as tetrahydrofurfuryl alcohol PEG ether (glycofurol, availablecommercially from BASF under the trade name Tetraglycol) or methoxy PEG(Union Carbide); amides, such as 2-pyrrolidone, 2-piperidone,caprolactam, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone,N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide, andpolyvinypyrrolidone; esters, such as ethyl propionate,tributylcitrate,acetyl triethylcitrate, acetyl tributyl citrate,triethylcitrate, ethyl oleate, ethyl caprylate, ethyl butyrate,triacetin, propylene glycol monoacetate, propylene glycol diacetate,caprolactone and isomers thereof, valerolactone and isomers thereof,butyrolactone and isomers thereof; and other solubilizers known in theart, such as dimethyl acetamide, dimethyl isosorbide (Arlasolve DMI(ICI)), N-methyl pyrrolidones (Pharmasolve (ISP)), monooctanoin,diethylene glycol nonoethyl ether (available from Gattefosse under thetrade name Transcutol), and water. Mixtures of solubilizers are alsowithin the scope of the invention.

Except as indicated, compounds mentioned herein are readily availablefrom standard commercial sources.

The clear liquid composition is visually clear to the unaided eye, as itwill contain less than 5%, less than 3% or less than 1% by wt. ofsuspended solids based upon the total weight of the composition.

Although not necessary, a composition or kit of the present inventionmay include a chelating agent, preservative, antioxidant, adsorbents,acidifying agent, alkalizing agent, antifoaming agent, buffering agent,colorant, electrolyte, salt, stabilizer, tonicity modifier, diluent,other pharmaceutical excipient, or a combination thereof.

As used herein, the term “antioxidant” is intended to mean an agent thatinhibits oxidation and is thus used to prevent the deterioration ofpreparations by the oxidative process. Such compounds include, by way ofexample and without limitation, ascorbic acid, ascorbic palmitate,Vitamin E, Vitamin E derivative, butylated hydroxyanisole, butylatedhydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate,sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate,sodium metalbisulfite and other such materials known to those ofordinary skill in the art.

As used herein, the term chelating agent is intended to mean a compoundthat chelates metal ions in solution. Exemplary chelating agents includeEDTA (tetrasodium ethylenediaminetetraacetate), DTPA (pentasodiumdiethylenetriaminepentaacetate), HEDTA (trisodium salt ofN-(hydroxyethyl)-ethylene-diaminetriacetic acid), NTA (trisodiumnitrilotriacetate), disodium ethanoldiglycine (Na₂EDG), sodiumdiethanolglycine (DEGNa), citric acid, and other compounds known tothose of ordinary skill in the art.

As used herein, the term “adsorbent” is intended to mean an agentcapable of holding other molecules onto its surface by physical orchemical (chemisorption) means. Such compounds include, by way ofexample and without limitation, powdered and activated charcoal andother materials known to one of ordinary skill in the art.

As used herein, the term “alkalizing agent” is intended to mean acompound used to provide an alkaline medium. Such compounds include, byway of example and without limitation, ammonia solution, ammoniumcarbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodiumborate, sodium carbonate, sodium bicarbonate, sodium hydroxide,triethanolamine, and trolamine and others known to those of ordinaryskill in the art.

As used herein, the term “acidifying agent” is intended to mean acompound used to provide an acidic medium. Such compounds include, byway of example and without limitation, acetic acid, amino acid, citricacid, fumaric acid and other alpha-hydroxy acids, hydrochloric acid,ascorbic acid, and nitric acid and others known to those of ordinaryskill in the art.

As used herein, the term “antifoaming agent” is intended to mean acompound or compounds that prevents or reduces the amount of foamingthat forms on the surface of the fill composition. Suitable antifoamingagents include by way of example and without limitation, dimethicone,SIMETHICONE, octoxynol and others known to those of ordinary skill inthe art.

As used herein, the term “buffering agent” is intended to mean acompound used to resist a change in pH upon dilution or addition of acidor alkali. Such compounds include, by way of example and withoutlimitation, potassium metaphosphate, potassium phosphate, monobasicsodium acetate and sodium citrate anhydrous and dehydrate and other suchmaterials known to those of ordinary skill in the art.

As used herein, the term “diluent” or “filler” is intended to mean inertsubstances used as fillers to create the desired bulk, flow properties,and compression characteristics in the preparation of tablets andcapsules. Such compounds include, by way of example and withoutlimitation, dibasic calcium phosphate, kaolin, lactose, sucrose,mannitol, microcrystalline cellulose, powdered cellulose, precipitatedcalcium carbonate, sorbitol, and starch and other materials known to oneof ordinary skill in the art.

As used herein, the term “preservative” is intended to mean a compoundused to prevent the growth of microorganisms. Such compounds include, byway of example and without limitation, benzalkonium chloride,benzethonium chloride, benzoic acid, benzyl alcohol, cetylpyridiniumchloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuricnitrate, phenylmercuric acetate, thimerosal, metacresol, myristylgammapicolinium chloride, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, sorbic acid, thymol, and methyl, ethyl,propyl, or butyl parabens and others known to those of ordinary skill inthe art.

As used herein, the term “colorant” is intended to mean a compound usedto impart color to pharmaceutical preparations. Such compounds include,by way of example and without limitation, FD&C Red No. 3, FD&C Red No.20, FD&C Yellow No. 6, FD&C Blue No. 2, FD&C Green No. 5, FD&C OrangeNo. 5, FD&C Red No. 8, caramel, and iron oxide (black, red, yellow),other FD&C dyes and natural coloring agents such as grape skin extract,beet red powder, beta-carotene, annato, carmine, turmeric, paprika,combinations thereof and other such materials known to those of ordinaryskill in the art.

As used herein, the term “stabilizer” is intended to mean a compoundused to stabilize an active agent against physical, chemical, orbiochemical processes that would otherwise reduce the therapeuticactivity of the agent. Suitable stabilizers include, by way of exampleand without limitation, albumin, sialic acid, creatinine, glycine andother amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide,sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethyleneglycols, sodium caprylate and sodium saccharin and others known to thoseof ordinary skill in the art.

As used herein, the term “tonicity modifier” is intended to mean acompound or compounds that can be used to adjust the tonicity of theliquid formulation. Suitable tonicity modifiers include glycerin,lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol,trehalose and others known to those or ordinary skill in the art.

The composition of the invention can also include oils such as fixedoils, peanut oil, sesame oil, cottonseed oil, corn oil and olive oil;fatty acids such as oleic acid, stearic acid and isostearic acid; andfatty acid esters such as ethyl oleate, isopropyl myristate, fatty acidglycerides and acetylated fatty acid glycerides. The composition canalso include alcohol such as ethanol, isopropanol, hexadecyl alcohol,glycerol and propylene glycol; glycerol ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol; ethers such as poly(ethyleneglycol) 450; petroleum hydrocarbons such as mineral oil and petrolatum;water; a pharmaceutically suitable surfactant, suspending agent oremulsifying agent; or mixtures thereof.

It should be understood that the compounds used in the art ofpharmaceutical formulation generally serve a variety of functions orpurposes. Thus, if a compound named herein is mentioned only once or isused to define more than one term herein, its purpose or function shouldnot be construed as being limited solely to that named purpose(s) orfunction(s).

One or more of the components of the formulation can be present in itsfree base or pharmaceutically or analytically acceptable salt form. Asused herein, “pharmaceutically or analytically acceptable salt” refersto a compound that has been modified by reacting it with an acid or baseas needed to form an ionically bound pair. Examples of acceptable saltsinclude conventional non-toxic salts formed, for example, from non-toxicinorganic or organic acids. Suitable non-toxic salts include thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfonic, sulfamic, phosphoric, nitric and others known tothose of ordinary skill in the art. The salts prepared from organicacids such as amino acids, acetic, propionic, succinic, glycolic,stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and others known to those of ordinaryskill in the art. Lists of other suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th). ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, the relevant disclosure of which is herebyincorporated by reference.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith tissues of human beings and animals and without excessive toxicity,irritation, allergic response, or any other problem or complication,commensurate with a reasonable benefit/risk ratio.

A dosage form can be made by any conventional means known in thepharmaceutical industry. A liquid dosage form can be prepared byproviding at least one liquid carrier and said improved composition in acontainer. One or more other excipients can be included in the liquiddosage form. A solid dosage form can be prepared by providing at leastone solid carrier and said improved composition. One or more otherexcipients can be included in the solid dosage form.

A dosage form can be packaged using conventional packaging equipment andmaterials. It can be included in a pack, bottle, via, bag, syringe,envelope, packet, blister pack, box, ampoule, or other such container.

The invention includes a method for improving the clinical status of astatistically significant number of subjects of in a population ofsubjects having a neurological condition, the method comprising:administering to the population of subjects a neuroprotectivecomposition; and determining the clinical status of the subjects toestablish the improved clinical status. In some embodiments, thestatistically significant number is at least 5%, at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80% or at least 90% of the population. In someembodiments, the neuroprotective composition comprises one or more otherpharmacologically active compounds.

As used herein a “derivative” is: a) a chemical substance that isrelated structurally to a first chemical substance and theoreticallyderivable from it; b) a compound that is formed from a similar firstcompound or a compound that can be imagined to arise from another firstcompound, if one atom of the first compound is replaced with anotheratom or group of atoms; c) a compound derived or obtained from a parentcompound and containing essential elements of the parent compound; or d)a chemical compound that may be produced from first compound of similarstructure in one or more steps. For example, a derivative may include adeuterated form, oxidized form, dehydrated, unsaturated, polymerconjugated or glycosilated form thereof or may include an ester, amide,lactone, homolog, ether, thioether, cyano, amino, alkylamino,sulfhydryl, heterocyclic, heterocyclic ring-fused, polymerized,pegylated, benzylidenyl, triazolyl, piperazinyl or deuterated formthereof.

In view of the above description and the examples below, one of ordinaryskill in the art will be able to practice the invention as claimedwithout undue experimentation. The foregoing will be better understoodwith reference to the following examples that detail certain proceduresfor the preparation of embodiments of the present invention. Allreferences made to these examples are for the purposes of illustration.The following examples should not be considered exhaustive, but merelyillustrative of only a few of the many embodiments contemplated by thepresent invention.

The triterpenes can be purchased from Sigma Chemical Co. (St. Louis,Mo.).

Example 1 Preparation of Triterpene Mixtures

The following compositions were made by mixing the specified triterpenesin the approximate molar ratios indicated. No specific order of mixingis required. The triterpenes can be mixed in any form in the presence orabsence of liquid.

Triterpene (Approximate Relative Molar Content) Oleanolic UrsolicBetulinic Composition acid (O) acid (U) acid (B) I (A-C) 3 2.2 1 II(A-C) 7.8 7.4 1 III (A-C) 10 1 1 IV (A-C) 1 10 1 V (A-C) 1 1 10 VI (A-C)1 1 0 VII (A-C) 1 1 1 VIII (A-C) 10 1 0 IX (A-C) 1 10 0

For each composition, three different respective solutions were made,whereby the total concentration of triterpenes in each solution wasapproximately 9 μM, 18 μM, or 36 μM.

Composition Triterpene (Approximate Content of Each, μM) (totaltriterpene Oleanolic Ursolic Betulinic content, μM) acid (O) acid (U)acid (B) I-A (36) 17.4 12.8 5.8 I-B (18) 8.7 6.4 2.9 I-C (9) 4.4 3.2 1.5II-A (36) 17.3 16.4 2.2 II-B (18) 8.7 8.2 1.1 II-C (9) 4.3 4.1 0.6 III-A(36) 30 3 3 III-B (18) 15 1.5 1.5 III-C (9) 7.5 0.75 0.75 IV-A (36) 3 303 IV-B (18) 1.5 15 1.5 IV-C (9) 0.75 7.5 0.75 V-A (36) 3 3 30 V-B (18)1.5 1.5 15 V-C (9) 0.75 0.75 7.5 VI-A (36) 18 18 0 VI-B (18) 9 9 0 VI-C(9) 4.5 4.5 0 VII-A (36) 12 12 12 VII-B (18) 6 6 6 VII-C (9) 3 3 3VIII-A (36) 32.7 3.3 0 VIII-B (18) 16.35 1.65 0 VIII-C (9) 8.2 0.8 0IX-A (36) 3.3 32.7 0 IX-B (18) 1.65 16.35 0 IX-C (9) 0.8 8.2 0

Example 2 Preparation of Improved Pharmaceutical Compositions

Improved compositions can be prepared by mixing the individualtriterpene components thereof to form a mixture. The triterpene mixturesprepared above that provided efficacy were formulated into improvedpharmaceutical compositions.

Improved Composition with Oleanolic Acid and Ursolic Acid

Known amounts of oleanolic acid and ursolic acid were mixed according toa predetermined molar ratio of the components as defined herein. Thecomponents were mixed in solid form or were mixed in solvent(s), e.g.methanol, ethanol, chloroform, acetone, propanol, dimethyl sulfoxide(DMSO), dimethylformamide (DMF), dimethylacetamide (DMAC),N-methylpyrrolidone (NMP), water or mixtures thereof. The resultantmixture contained the components in the relative molar ratios asdescribed herein.

For a pharmaceutically acceptable improved composition, at least onepharmaceutically acceptable excipient was mixed in with thepharmacologically active agents. An improved composition is formulatedfor administration to a mammal.

Improved Composition with Oleanolic Acid and Betulinic Acid

Known amounts of oleanolic acid and betulinic acid were mixed accordingto a predetermined molar ratio of the components as defined herein. Thecomponents were mixed in solid form or were mixed in solvent(s), e.g.methanol, ethanol, chloroform, acetone, propanol, dimethyl sulfoxide(DMSO), dimethylformamide (DMF), dimethylacetamide (DMAC),N-methylpyrrolidone (NMP), water or mixtures thereof. The resultantmixture contained the components in the relative molar ratios asdescribed herein.

For a pharmaceutically acceptable improved composition, at least onepharmaceutically acceptable excipient was mixed in with thepharmacologically active agents. An improved composition is formulatedfor administration to a mammal.

Improved Composition with Oleanolic Acid, Ursolic Acid and BetulinicAcid

Known amounts of oleanolic acid, ursolic acid and betulinic acid weremixed according to a predetermined molar ratio of the components asdefined herein. The components were mixed in solid form or were mixed insolvent(s), e.g. methanol, ethanol, chloroform, acetone, propanol,dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide(DMAC), N-methylpyrrolidone (NMP), water or mixtures thereof. Theresultant mixture contained the components in the relative molar ratiosas described herein.

For a pharmaceutically acceptable improved composition, at least onepharmaceutically acceptable excipient was mixed in with thepharmacologically active agents. An improved composition is formulatedfor administration to a mammal.

Example 3 Evaluation of Compositions in Brain Slice-Based OGD Assay

Coronal brain slices (250 m thick) were prepared from postnatal day 10Sprague-Dawley rat pups of either gender (Charles River) and establishedin organotypic culture. Animals were sacrificed in accordance with NIHguidelines and under Duke IACUC approval and oversight. Briefly, braintissue slices were cut in ice-cold artificial cerebrospinal fluid (ACSF)and plated in interface configuration on top of culture medium(Neurobasal A medium supplemented with 15% heat-inactivated horse serum,10 mM KCl, 10 mM HEPES, 100 U/ml penicillin/streptomycin, 1 mM sodiumpyruvate, and 1 mM L-glutamine) set in 0.5% reagent-grade agarose. Tomodel ischemic injury, brain slices were subjected to oxygen-glucosedeprivation (OGD) by exposure to glucose-free, N₂-bubbled ACSFcontaining low 02 (<0.5%) for 5.5 min.

One hour later, control and OGD-treated brain slices were biolisticallytransfected with DNAs encoding yellow fluorescent protein (YFP). Forassays modeling neurodegeneration in AD or FTD, brain slices wereco-transfected with YFP together with an expression construct to WTamyloid precursor protein (APP), or with YFP together with a cDNAconstructed in house encoded human Tau4RON (identical to NCBI ReferenceSequence NM_016834), respectively. Brain slice explants were thenincubated for 24 h under 5% CO₂ at 37° C. for OGD assays; or for 3 d forAPP- and tau4RON-induced neurodegeneration assays. Compositions wereadded at the indicated concentrations to culture medium at the time ofbrain slice explantation.

For all brain slice assays, numbers of healthy pyramidal neurons in thecortical regions of each brain slice were imaged on a Leica MZIIIFLfluorescence stereomicroscope. Cortical pyramidal neurons were readilyidentified by their characteristic positions and orientations in thecortical plate, and by their prominent extension of a single, apicaldendrite radially towards the pial surface. Healthy cortical pyramidalneurons were deemed as those 1) presenting a stout and brightly labeledcell body located within the pyramidal neuronal layers of the cortex; 2)retaining a clear apical dendrite extending radially towards the pialsurface the slice; 3) expressing ≥2 clear basal dendrites ≥2 cell bodydiameters long directly from the neuronal soma; and 4) showing clear andcontinuous cytoplasmic labeling with the YFP visual marker in the somaand throughout all neuronal processes. Statistically significantdifferences with respect to the negative control condition (OGD,APP-transfected, or tau4R-transfected treated with DMSO carrier only)were determined using ANOVA followed by Dunnett's post hoc comparisontest at the 0.05 confidence level, with N=12 brain slices per condition.Each experiment was carried out at least 2 times.

Example 4 Time-Delay Brain-Slice Assay for Determination ofNeuroprotection

This assay was conducted according to Example 3 except that thefollowing changes were made. A specified length of time was allowedbetween OGD and introduction of a composition being evaluated. Theability of the composition to provide neuroprotection to brain slices iftreatment was delayed relative to the timing of the OGD treatment wasdetermined.

Example 5 Evaluation of Compositions for Nrf2 Activation and ARE GeneExpression

Nrf2 Activation

Primary corticostriatal neuronal co-cultures were prepared from E18Sprague-Dawley rat or C57Bl/6 mouse embryos of either gender. Forluciferase reporter assays, the Cignal Antioxidant Response Reporter kit(Qiagen) was used. The 5×ARE luciferase reporter mixture at 40:1luciferase:Renilla plasmid was transfected into cortical and striatalneurons separately using an Amaxa electroporation device (Lonza). Afterelectroporation, neurons were pooled and immediately plated into 96-wellplates containing mature glial cultures. After culturing for 96 h,compositions were added at the indicated concentrations for 7 or 24 hprior to harvesting using Dual-Glo Luciferase Assay System protocol andreagents (Promega). Dual-wavelength luminescence was detected using aSpectraMax L microplate reader (Molecular Devices). Luciferase valueswere normalized to the internal Renilla control and fold-expression overthe DMSO-only treatment control was calculated. At least 3 independentexperiments were done using 4-6 biological replicates.

ARE Gene Expression

For qPCR quantification of ARE target gene expression levels, corticaland striatal neurons were plated onto 96-well plates containing matureglial cultures and cultured for 96 h. Composition was added to culturesat the indicated concentrations for 6 h. At the end of the treatmentperiod, cells were lysed and total RNA was isolated using Absolutely RNAmini-prep kits (Agilent Technologies/Stratagene). cDNA was generatedusing oligo dT primers and Superscript II reverse transcriptase(Invitrogen). Resulting cDNA was used for quantitative PCR of genetranscripts using SYBR Green Real-Time PCR Master Mix (LifeTechnologies) and the following mouse primers, for: Gclc

(SEQ ID NO. 1) (forward-5′ TGGCCACTATCTGCCCAATT-3′and

(SEQ ID NO. 2) reverse-5′-GTCTGACACGTAGCCTCGGTAA-3′),Nqo1

(SEQ ID NO. 3) (forward-5′-GCCCGCATGCAGATCCT-3′and

(SEQ ID NO. 4) reverse 5′-GGTCTCCTCCCAGACGGTTT3′),Srx

(SEQ ID NO. 5) (forward-5′-GCTTCCTCTCGGGAGTCCTT-3′and

(SEQ ID NO. 6) reverse-5′-CAGCAACAGCGACTACGAAGTAA-3′),and Hmox1

(SEQ ID NO. 7) (forward-5′-CCTCACTGGCAGGAAATCATC-3′and

(SEQ ID NO. 8) reverse-5′-CCTCGTGGAGACGCTTTACATA-3′)(Integrated DNA Technologies). For rat corticostriatal co-culturesamples, qPCR primers used were as previously described (van Roon-Mom,W. M. et al. Mutant huntingtin activates Nrf2-responsive genes andimpairs dopamine synthesis in a PC12 model of Huntington's disease. BMCMolecular Biology (2008), 9, 1-13, doi:10.1186/1471-2199-9-84). Eachbiological sample was measured in triplicate on a ViiA 7 real-time PCRinstrument (Applied Biosystems); fold expression was calculated afternormalization to corresponding control GAPDH levels.

Example 6 Evaluation of a Composition in an In Vitro CorticostriatalCo-Culture Assay for Huntington's Disease

In this assay, instead of using intact brain slices, mutant htt isintroduced via electroporation into high-density, mixed co-cultures ofcortical neurons, striatal neurons, and glia arrayed in 96-well plates.The goal of this assay platform is to combine the biological/clinicalrelevance of a complex primary culture system that recapitulates keyaspects of the interconnectivity of disease-relevant neuronalpopulations in vivo, with the ability to conduct large-scale fullyautomated screening campaigns. In this assay, over the course of 1-2weeks in vitro, transfected mutant htt constructs induce the progressivedegeneration of both striatal and cortical neurons that are subsequentlyquantified using automated image acquisition and object detectionalgorithms on the Cellomics Arrayscan VTI platform. Each data point wasdrawn from 6 wells with 16 images in each well automatically captured,processed, and analyzed on the Cellomics Arrayscan using protocolsdeveloped during a large-scale screening campaign being conducted inassociation with the Cure Huntington's Disease Initiative. In a fullrun, some 25,000 images are collected and analyzed in each cycle, 4cycles per week.

Cortico-Striatal Co-Culture Assay Platform.

Pure glial cultures are prepared in advance of neuronal plating toestablish 96-well plates with confluent glial beds. Cortical andstriatal tissue are then dissociated separately and “nucleofected” withappropriate DNA constructs and are distinguishable later by theexpression of different fluorescent proteins such as YFP, CFP, andmCherry. These separately transfected cortical and striatal neurons arethen mixed thoroughly and plated into the 96-well plates containing thepreviously plated glial monolayers. Compositions are tested in thiscortico-striatal co-culture platform.

Example 7 Treatment of Neurological Condition Including but not Limitedto Alzheimer's Disease

Method A. Neuroprotective Composition Therapy

A subject presenting with Alzheimer's disease is prescribedneuroprotective composition, and therapeutically relevant doses areadministered to the subject according to a prescribed dosing regimen fora period of time. The subject's level of therapeutic response isdetermined periodically. If the level of therapeutic response is too lowat one dose, then the dose is escalated according to a predetermineddose escalation schedule until the desired level of therapeutic responsein the subject is achieved. Treatment of the subject with theneuroprotective composition is continued as needed and the dose ordosing regimen can be adjusted as needed until the patient reaches thedesired clinical endpoint.

Method B. Combination Therapy: Neuroprotective Composition and AnotherTherapeutic Agent

Method A, above, is followed except that the subject is prescribed andadministered neuroprotective composition in combination with one or moreother therapeutic agents for the treatment of Alzheimer's disease, orsymptoms thereof. Then one or more other therapeutic agents can beadministered before, after or with the neuroprotective composition. Doseescalation (or de-escalation) of the one or more other therapeuticagents can also be done. Suitable one or more other therapeutic agentsinclude Namenda™ (memantine HCl), Aricept™ (donepezil), Razadyne™(galantamine), Exelon™ (rivastigmine), Cognex™ (tacrine), andamantadine.

Example 8 Treatment of Neurological Condition Including but not Limitedto Huntington's Disease

Method A. Neuroprotective Composition Therapy

A subject presenting with Huntington's disease is prescribed theneuroprotective composition, and therapeutically relevant doses areadministered to the subject according to a prescribed dosing regimen fora period of time. The subject's level of therapeutic response isdetermined periodically. If the level of therapeutic response is too lowat one dose, then the dose is escalated according to a predetermineddose escalation schedule until the desired level of therapeutic responsein the subject is achieved. Treatment of the subject withneuroprotective composition is continued as needed and the dose ordosing regimen can be adjusted as needed until the patient reaches thedesired clinical endpoint. The doses administered can be similar tothose described herein.

Method B. Combination Therapy: Neuroprotective Composition and AnotherTherapeutic Agent

Method A, above, is followed except that the subject is prescribed andadministered neuroprotective composition in combination with one or moreother therapeutic agents for the treatment of Huntington's disease, orsymptoms thereof. The one or more other therapeutic agents can beadministered before, after or with the neuroprotective composition. Doseescalation (or de-escalation) of the one or more other therapeuticagents can also be done. Suitable one or more other therapeutic agentsinclude Vitamin E, Baclofen (a derivative of CoQ10), Lamotrigine (ananticonvulsant), remacemide (an anesthetic which is a low affinity NMDAantagonist), and riluzole (Na channel blocker).

Example 9 Treatment of Neurological Condition Including but not Limitedto Ischemic Stroke

Method A. Neuroprotective Composition Therapy

A subject presenting with ischemic stroke is prescribed theneuroprotective composition, and therapeutically relevant doses areadministered to the subject according to a prescribed dosing regimen fora period of time. The subject's level of therapeutic response isdetermined periodically. If the level of therapeutic response is too lowat one dose, then the dose is escalated according to a predetermineddose escalation schedule until the desired level of therapeutic responsein the subject is achieved. Treatment of the subject with theneuroprotective composition is continued as needed and the dose ordosing regimen can be adjusted as needed until the patient reaches thedesired clinical endpoint. The doses administered can be similar tothose described herein.

Method B. Combination Therapy: Neuroprotective Composition and AnotherTherapeutic Agent

Method A, above, is followed except that the subject is prescribed andadministered neuroprotective composition in combination with one or moreother therapeutic agents for the treatment of ischemic stroke, orsymptoms thereof. The one or more other therapeutic agents can beadministered before, after or with the neuroprotective composition. Doseescalation (or de-escalation) of the one or more other therapeuticagents can also be done.

Example 10 Treatment of Neurological Condition Including but not Limitedto Parkinson's Disease

Method A. Neuroprotective Composition Therapy

A subject presenting with Parkinson's disease is prescribed theneuroprotective composition, and therapeutically relevant doses areadministered to the subject according to a prescribed dosing regimen fora period of time. The subject's level of therapeutic response isdetermined periodically. If the level of therapeutic response is too lowat one dose, then the dose is escalated according to a predetermineddose escalation schedule until the desired level of therapeutic responsein the subject is achieved. Treatment of the subject withneuroprotective composition is continued as needed and the dose ordosing regimen can be adjusted as needed until the patient reaches thedesired clinical endpoint. The doses administered can be similar tothose described herein.

Method B. Combination Therapy: Neuroprotective Composition and AnotherTherapeutic Agent

Method A, above, is followed except that the subject is prescribed andadministered neuroprotective composition in combination with one or moreother therapeutic agents for the treatment of Parkinson's disease, orsymptoms thereof. The one or more other therapeutic agents can beadministered before, after or with the neuroprotective composition. Doseescalation (or de-escalation) of the one or more other therapeuticagents can also be done. Suitable one or more other therapeutic agentsinclude a combination of carbidopa and levodopa, rasagiline,pramipexole, ropinrole, amantadine, memantine, entacapone, rotigotine,benztropine, selegiline, biperiden, a combination of carbidopa andlevodopa and entacapone, trihexylphenidyl, rivastigmine, apomorphine,levodopa, carbidopa, bromocriptine, belladonna, tolcapone, or acombination thereof.

Example 11 Evaluation of Neuroprotective Composition in an In VitroAssay for Alzheimer's Disease (tau4R and APP)

In the rat brain slice model for APP/Abeta-induced degeneration ofcortical pyramidal neurons biolistic transfection is used not only tointroduce vital markers such as YFP, but also to introduce disease geneconstructs into the same neuronal populations in the brain slices. Thus,the APP/Aβ brain slice model co-transfects YFP with APP isoforms,leading to the progressive degeneration of cortical pyramidal neuronsover the course of 3-4 days after brain slice preparation andtransfection. The data demonstrate that the neuroprotective compositionis to provide concentration-dependent neuroprotection to APP-transfectedbrain slices.

Example 12 Evaluation of Neuroprotective Composition in an In VitroAssay for Stroke and Non-Stroke

Method A. Stroke: Preparation of Cortical Brain Slices and OGD.

Neocortical brain slices were prepared from PND 7 Sprague-Dawley ratpups. The cerebral cortex was dissected, cut into 400-μ-thick slices andtransferred into a container containing cold artificial cerebrospinalfluid with 1 μM MK-801 before plating; MK-801 was not included in anysubsequent procedures. To mimic ischemic injury using transientoxygen-glucose deprivation (OGD), slices from one hemisphere of eachbrain were exposed to glucose-free, N₂-bubbled artificial cerebrospinalfluid for 7.5 min in a low 02 (0.5%) environment. The OGD slices werethen plated side-by-side with control slices from the contralateralhemisphere on nitrocellulose or Millicell (Millipore) permeablemembranes, which were prepared identically except for no OGD. Thirtyminutes after plating, the brain slice pairs were transfected,transferred to 24-well plates, and incubated at 37° C. under 5% CO₂ inhumidified chambers. In each experiment, 5-6 minutes of oxygen-glucosedeprivation (OGD) was used to induce >50% loss of healthy corticalneurons by 24 hrs. A set concentration of control material used as theinternal positive control. Various concentrations of the compositionsare evaluated

Method B. Non-Stroke Brain Slice Assay.

Compositions were tested on “nonstroked” brain slices; that is, onesthat were sliced and transfected with YFP but not subjected toadditional trauma via OGD. See experimental procedure outlined above.

Example 13 Preparation of Dosage Forms Containing Triterpene-BasedComposition

Method A. Cremophor-Based Drug Delivery System

The following ingredients are provided in the amounts indicated.

Percent of Reagent Name Function Formulation (% w/w) PBI-01011 Activeagent 3.7 Vitamin E Antioxidant 0.1 Labrasol Surfactant 9.2 EthanolCo-solvent 9.6 Cremophor EL Surfactant 62.6 Cremophor RH40 Surfactant14.7

The excipients are dispensed into a jar and shook in a New BrunswickScientific C24KC Refrigerated Incubator shaker for 24 hours at 60° C. toensure homogeneity. The samples are then pulled and visually inspectedfor solubilization.

Method B. GMO/Cremophor-Based Drug Delivery System

The following ingredients are provided in the amounts indicated.

Percent of Reagent Name Function Formulation (% w/w) PBI-01011 Activeagent 4.7 Vitamin E Antioxidant 0.1 Labrasol Surfactant 8.5 EthanolCo-solvent 7.6 Cremophor EL Surfactant 56.1 Glycerol MonooleateSurfactant 23.2

The procedure of Method A is followed.

Method C. Labrasol-Based Drug Delivery System

The following ingredients are provided in the amounts indicated.

Percent of Reagent Name Function Formulation (% w/w) PBI-01011 Activeagent 3.7 Vitamin E Antioxidant 0.1 Labrasol Surfactant 86.6 EthanolCo-solvent 9.6

The procedure of Method A is followed.

Method D. Vitamin E-TPGS Based Micelle Forming System

The following ingredients are provided in the amounts indicated.

Component Function Weight % (w/w) Vitamin E Antioxidant 1.0 Vitamin ETPGS Surfactant 95.2 PBI-01011 Active agent 3.8

The procedure of Method A is followed.

Method E. Multi-Component Drug Delivery System

The following ingredients e a provided in the amounts indicated.

Component Weight (g) Weight % (w/w) Vitamin E 10.0 1.0 Cremophor ELP580.4 55.9 Labrasol 89.0 8.6 Glycerol Monooleate 241.0 23.2 Ethanol 80.07.7 PBI-01011 38.5 3.7 Total 1038.9 100

The procedure of Method A is followed.

Example 14 Preparation of Enteric Coated Capsules

Step I: Preparation of Liquid-Filled Capsule

Hard gelatin capsules (50 counts, 00 size) are filled with a liquidcomposition of Example 13. These capsules are filled with 800 mg of theformulation and then sealed by hand with a 50% ethanol/50% watersolution. The capsules are then banded by hand with 22% gelatin solutioncontaining the following ingredients in the amounts indicated.

Ingredient Wt. (g) Gelatin 140.0 Polysorbate 80 6.0 Water 454.0 Total650.0

The gelatin solution mixed thoroughly and allowed to swell for 1-2hours. After the swelling period, the solution is covered tightly andplaced in a 55° C. oven and allowed to liquefy. Once the entire gelatinsolution is liquid, the banding is performed. Using a pointed round 3/0artist brush, the gelatin solution is painted onto the capsules. Bandingkit provided by Shionogi is used. After the banding, the capsules arekept at ambient conditions for 12 hours to allow the band to cure.

Step II: Coating of Liquid-Filled Capsule

A coating dispersion is prepared from the ingredients listed in thetable below.

Ingredient Wt. % Solids % Solids (g) g/Batch Eudragit L30D55 40.4 60.576.5 254.9 TEC 1.8 9.0 11.4 11.4 AlTalc 500V 6.1 30.5 38.5 38.5 Water51.7 na na 326.2 Total 100.0 100.0 126.4 631.0

If banded capsules according to Step I are used, the dispersion isapplied to the capsules to a 20.0 mg/cm² coating level. The followingconditions are used to coat the capsules.

Parameters Set-up Coating Equipment Vector LDCS-3 Batch Size 500 g InletAir Temp. 40° C. Exhaust Air Temp. 27-30° C. Inlet Air Volume 20-25 CFMPan Speed 20 rpm Pump Speed 9 rpm (3.5 to 4.0 g/min) Nozzle Pressure 15psi Nozzle diameter 1.0 mm Distance from tablet bed* 2-3 in *Spraynozzle was set such that both the nozzle and spray path were under theflow path of inlet air.

Example 15 Preparation of a Tablet Comprising Triterpene-BasedComposition

An initial tabletting mixture of 3% Syloid 244FP and 97%microcrystalline cellulose (MCC) is mixed. Then, an existing batch ofcomposition prepared according to Example 13 is incorporated into theSyloid/MCC mixture via wet granulation. This mixture is labeled “InitialTabletting Mixture) in the table below. Additional MCC is addedextra-granularly to increase compressibility. This addition to theInitial Tabletting Mixture is labeled as “Extra-granular Addition.” Theresultant mixture from the extra-granular addition is the samecomposition as the “Final Tabletting Mixture.”

Component Weight (g) Weight % (w/w) Initial Tabletting MixtureMicrocrystalline cellulose 48.5 74.2 Colloidal Silicon Dioxide/Syloid1.5 2.3 244FP Formulation from Ex. 13 15.351 23.5 Total 65.351 100.0Extragranular addition Initial Tabulating Mixture 2.5 50.0Microcrystalline cellulose 2.5 50.0 Total 5 100.0 Final TablettingMixture: Abbreviated Microcrystalline cellulose 4.36 87.11 ColloidalSilicon Dioxide/Syloid 0.06 1.15 244FP Formulation from Ex. 13 0.5911.75 Total 5.00 100 Final Tabletting Mixture: Detailed Microcrystallinecellulose 4.36 87.11 Colloidal Silicon Dioxide/Syloid 0.06 1.15 244FPVitamin E 0.01 0.11 Cremophor ELP 0.33 6.56 Labrasol 0.05 1.01 GlycerolMonooleate 0.14 2.72 Ethanol 0.05 0.90 PBI-01011 0.02 0.44 Total 5.00100.00

Syloid 244FP is a colloidal silicon dioxide manufactured by GraceDavison. Colloidal silicon dioxide is commonly used to provide severalfunctions, such as an adsorbant, glidant, and tablet disintegrant.Syloid 244FP was chosen for its ability to adsorb 3 times its weight inoil and for its 5.5 micron particle size.

Example 16 Determining Efficacy of Triterpene-Based Compositions inTreating Autoimmune Condition, Disease or Disorder

The method of Martin et al. (“Natural triterpenes modulateimmune-inflammatory markers of experimental autoimmuneencephalomyelitis: therapeutic implications of multiple sclerosis” inBrit. J. Pharmacol. (2012), 166, 1708-1723, the entire disclosure ofwhich is hereby incorporated by reference) except that compositions ofthe invention comprising at least two or at least three triterpenes areevaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.33-5:0.33-5.

Example 17 Determining Efficacy of Triterpene-Based Compositions inTreating Tuberculosis

The method of Jimenez-Arellanes et al. (“Ursolic and oleanolic acids asantimicrobial and immunomodulatory compounds for tuberculosis treatment”in BMC Complementary and Altern. Med. (2013), 13, 258-269, the entiredisclosure of which is hereby incorporated by reference) except thatcompositions of the invention comprising at least two or at least threetriterpenes are evaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.33-5:0.33-5.

Example 18 Determining Efficacy of Triterpene-Based Compositions inTreating Cell-Proliferation Related Condition, Disease or Disorder

Compositions of the invention are evaluated for treating cellproliferation condition, disease or disorder according to any of themethods of Newman et al. (U.S. Pat. Nos. 8,187,644B2, 8,394,434B2,7,402,325B2, 9,494,589B2, 9,846,156B2, 8,367,363B2, the entiredisclosures of which are hereby incorporated by reference) or of Cauniet al. (“Effects of ursolic and oleanolic acid on SK-MEL-2 melanomacells: in vitro and in vivo assays”, in Inter. J. Oncol. (2017), 51,1651-1660, the entire disclosure of which is hereby incorporated byreference) except that compositions of the invention comprising at leasttwo or at least three triterpenes and excluding cardiac glycoside areevaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.33-5:0.33-5.

Example 19 Determining Efficacy of Triterpene-Based Compositions inTreating Viral Infection

Compositions of the invention are evaluated for treating viralinfections listed above according to the methods of Newman et al. (WO2018/053123A1, the entire disclosure of which is hereby incorporated byreference) except that compositions of the invention comprising at leasttwo or at least three triterpenes and excluding cardiac glycoside areevaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.33-5:0.33-5.

Example 20 Determining Efficacy of Triterpene-Based Compositions inTreating Bacterial Infection

Compositions of the invention are evaluated for treating bacterialinfections listed above according to the methods of Kim et al.(“Antimicrobial action of oleanolic acid on Listeria monocytogenes,Enterococcus faecium, and Enterococcus faecalis” in PLOS ONE,DOI:10.1371/Journal.pone.0118800, (2015), 1-11, the entire disclosure ofwhich is hereby incorporated by reference) except that compositions ofthe invention comprising at least two or at least three triterpenes areevaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.33-5:0.33-5.

Example 21 Determining Efficacy of Triterpene-Based Compositions inTreating Diabetes

Compositions of the invention are evaluated for treating diabetes andrelated conditions according to the methods of Lo et al. (“Developmentof betulinic acid as an agonist of TGR5 receptor using a new in vitroassay” in Drug Des. Dvlp. Ther. (2016), 10, 2669-2676, the entiredisclosure of which is hereby incorporated by reference) or the methodsof Yin (“Inhibitory effects and actions of pentacyclic triterpenes uponglycation” in Biomedicine (2015), 5(3), 1-8, the entire disclosure ofwhich is hereby incorporated by reference), except that compositions ofthe invention comprising at least two or at least three triterpenes areevaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.33-5:0.33-5.

Example 22 Determining Efficacy of Triterpene-Based Compositions inTreating Musculoskeletal Condition, Disease or Disorder

Compositions of the invention are evaluated for treating musculoskeletalcondition, disease or disorder, in particular age-related loss of musclemass, according to the methods of Ebert et al. (“Identification andSmall Molecule Inhibition of an Activating Transcription Factor 4(ATF4)-dependent Pathway to Age-related Skeletal Muscle Weakness andAtrophy” in J. Biol. Chem. (2015), 290(42), 25497-25511, the entiredisclosure of which is hereby incorporated by reference) or the methodsof Kunkel et al. (“mRNA expression signatures of human skeletal muscleatrophy identify a natural compound that increases muscle mass” in CellMetab. (2011), 13(6), 627-638, the entire disclosure of which is herebyincorporated by reference) except that compositions of the inventioncomprising at least two or at least three triterpenes are evaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.33-5:0.33-5.

Example 23 Determining Efficacy of Triterpene-Based Compositions inTreating Parasitic Infection

Compositions of the invention are evaluated for treating parasiticinfection according to the methods of López et al. (“Phytochemicalcomposition, antiparasitic and alpha-glucosidase inhibition activitiesfrom Pelliciera rhizophorae” in Chem. Cent. J. (2015), 9:53, 1-11, theentire disclosure of which is hereby incorporated by reference) exceptthat compositions of the invention comprising at least two or at leastthree triterpenes are evaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.15-2.5:0.15-2.5. A composition comprising twotriterpenes at the following molar ratio(s) may be found to be active:OA:UA is 9-12:0.33-5 or OA:BA is 9-12:0.33-5.

Example 24 Determining Efficacy of Triterpene-Based Compositions inTreating Protozoal Infection

Compositions of the invention are evaluated for treating protozoalaccording to the methods of Yamamoto et al. (“The effect of ursolic acidon Leishmania amazonensis is related to programmed cell death andpresents therapeutic potential in experimental cutaneous leishmaniasis”in PLOS ONE DOI:10.1371/journal.pone.0144946, (2015), 1-19, the entiredisclosure of which is hereby incorporated by reference) except thatcompositions of the invention comprising at least two or at least threetriterpenes are evaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.15-2.5:0.15-2.5. A composition comprising twotriterpenes at the following molar ratio(s) may be found to be active:OA:UA is 9-12:0.33-5 or OA:BA is 9-12:0.33-5.

Example 25 Determining Efficacy of Triterpene-Based Compositions inTreating Oxidative Stress-Related Condition, Disease or Disorder

Compositions of the invention are evaluated for treating oxidativestress-related condition, disease or disorder according to the methodsof Madlala et al. (“Changes in renal and oxidative status associatedwith hypotensive effects of oleanolic acid and related syntheticderivatives in experimental animals” in PLOS ONEDOI:10.1371/journal.pone.0128192, (2015), 1-20, the entire disclosure ofwhich is hereby incorporated by reference) except that compositions ofthe invention comprising at least two or at least three triterpenes areevaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.15-2.5:0.15-2.5. A composition comprising twotriterpenes at the following molar ratio(s) may be found to be active:OA:UA is 9-12:0.33-5 or OA:BA is 9-12:0.33-5.

Example 26 Determining Efficacy of Triterpene-Based Compositions inTreating Gastrointestinal Condition, Disease or Disorder

Compositions of the invention are evaluated for treatinggastrointestinal condition, disease or disorder according to the methodsof Dinh et al. (“Bardoxolone methyl prevents high-fat diet-induced coloninflammation in mice” in J. Histochem. Cytochem. (2016), 64(4), 237-255,the entire disclosure of which is hereby incorporated by reference)except that compositions of the invention comprising at least two or atleast three triterpenes are evaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.15-2.5:0.15-2.5. A composition comprising twotriterpenes at the following molar ratio(s) may be found to be active:OA:UA is 9-12:0.33-5 or OA:BA is 9-12:0.33-5.

Example 27 Determining Efficacy of Triterpene-Based Compositions inTreating Angiogenesis-Related Condition, Disease or Disorder

Compositions of the invention are evaluated for treatingangiogenesis-related condition, disease or disorder according to themethods of Saraswati et al. (“Ursolic acid inhibits tumor angiogenesisand induces apoptosis through mitochondrial-dependent pathway in Ehrlichascites carcinoma tumor” in Chem. Biol. Interact. (2013), 206(2),153-165, the entire disclosure of which is hereby incorporated byreference) except that compositions of the invention comprising at leasttwo or at least three triterpenes are evaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.15-2.5:0.15-2.5. A composition comprising twotriterpenes at the following molar ratio(s) may be found to be active:OA:UA is 9-12:0.33-5 or OA:BA is 9-12:0.33-5.

Example 28 Determining Efficacy of Triterpene-Based Compositions inTreating Cardiovascular Condition, Disease or Disorder

Compositions of the invention are evaluated for treating cardiovascularcondition, disease or disorder, in particular hypertension, according tothe methods of Steinkamp-Fenske et al. (“Reciprocal upregulation ofendothelial nitric-oxide synthase and NADPH oxidase by betulinic acid inhuman endothelial cells” in J. Pharmacol. Exper. Therap. (2007), 322(2),836-842, the entire disclosure of which is hereby incorporated byreference) except that compositions of the invention comprising at leasttwo or at least three triterpenes are evaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.15-2.5:0.15-2.5. A composition comprising twotriterpenes at the following molar ratio(s) may be found to be active:OA:UA is 9-12:0.33-5 or OA:BA is 9-12:0.33-5.

Example 29 Determining Efficacy of Triterpene-Based Compositions inTreating Hepatic Condition, Disease or Disorder

Compositions of the invention are evaluated for treating hepaticcondition, disease or disorder according to the methods of Yi et al.(“Betulinic acid prevents alcohol-induced liver damage by improving theantioxidant system in mice” in J. Vet. Sci. (2014), 15(1), 141-148, theentire disclosure of which is hereby incorporated by reference) exceptthat compositions of the invention comprising at least two or at leastthree triterpenes are evaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.15-2.5:0.15-2.5. A composition comprising twotriterpenes at the following molar ratio(s) may be found to be active:OA:UA is 9-12:0.33-5 or OA:BA is 9-12:0.33-5.

Example 30 Determining Efficacy of Triterpene-Based Compositions inTreating Bone-Related Condition, Disease or Disorder

Compositions of the invention are evaluated for treating bone-relatedcondition, disease or disorder according to the methods of Choi et al.(“Betulinic acid synergistically enhances BMP2-induced bone formationvia stimulating Smad 1/5/8 and p38 pathways” in J. Biomed. Sci. (2016),23:45, 1-9, the entire disclosure of which is hereby incorporated byreference) except that compositions of the invention comprising at leasttwo or at least three triterpenes are evaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.15-2.5:0.15-2.5. A composition comprising twotriterpenes at the following molar ratio(s) may be found to be active:OA:UA is 9-12:0.33-5 or OA:BA is 9-12:0.33-5.

Example 31 Determining Efficacy of Triterpene-Based Compositions inTreating Dermatological Condition, Disease or Disorder, in ParticularWound

Compositions of the invention are evaluated for treating dermatologicalcondition, disease or disorder according to the methods of Ebeling etal. (“From a traditional medicinal plant to a rational drug:understanding the clinically proven wound healing efficacy of birch barkextract” in PLOS ONE (2014), 9(1), e86147, the entire disclosure ofwhich is hereby incorporated by reference) except that compositions ofthe invention comprising at least two or at least three triterpenes areevaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.15-2.5:0.15-2.5. A composition comprising twotriterpenes at the following molar ratio(s) may be found to be active:OA:UA is 9-12:0.33-5 or OA:BA is 9-12:0.33-5.

Example 32 Determining Efficacy of Triterpene-Based Compositions inTreating Renal Condition, Disease or Disorder

Compositions of the invention are evaluated for treating renalcondition, disease or disorder according to the methods of Madlala etal. (“Changes in renal and oxidative status associated with hypotensiveeffects of oleanolic acid and related synthetic derivatives inexperimental animals” in PLOS ONE DOI:10.1371/journal.pone.0128192,(2015), 1-20, the entire disclosure of which is hereby incorporated byreference) except that compositions of the invention comprising at leasttwo or at least three triterpenes are evaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.15-2.5:0.15-2.5. A composition comprising twotriterpenes at the following molar ratio(s) may be found to be active:OA:UA is 9-12:0.33-5 or OA:BA is 9-12:0.33-5.

Example 33 Determining Efficacy of Triterpene-Based Compositions inTreating Metabolic Condition, Disease or Disorder

Compositions of the invention are evaluated for treating metaboliccondition, disease or disorder according to the methods of Kunkel et al.(“mRNA expression signatures of human skeletal muscle atrophy identify anatural compound that increases muscle mass” in Cell Metab. (2011),13(6), 627-638, the entire disclosure of which is hereby incorporated byreference) except that compositions of the invention comprising at leasttwo or at least three triterpenes are evaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.15-2.5:0.15-2.5. A composition comprising twotriterpenes at the following molar ratio(s) may be found to be active:OA:UA is 9-12:0.33-5 or OA:BA is 9-12:0.33-5.

Example 34 Determining Efficacy of Triterpene-Based Compositions inTreating Pulmonary Condition, Disease or Disorder

Compositions of the invention are evaluated for treating pulmonarycondition, disease or disorder according to the methods of Feng et al.(“Inhibition of human neutrophil elastase by pentacyclic triterpenes” inPLOS ONE (2013), 8(12), e82794, the entire disclosure of which is herebyincorporated by reference) except that compositions of the inventioncomprising at least two or at least three triterpenes are evaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.15-2.5:0.15-2.5. A composition comprising twotriterpenes at the following molar ratio(s) may be found to be active:OA:UA is 9-12:0.33-5 or OA:BA is 9-12:0.33-5.

Example 35 Determining Efficacy of Triterpene-Based Compositions inTreating Viral Infection

Compositions of the invention are evaluated for treating viralinfection, in particular Flaviviriade infection, Togaviridae infection,Paramyxoviridae infection, and Filoviridae infection, according to themethods of Phoenix Biotechnology, Inc. (WO 2018/053123A1, the entiredisclosure of which is hereby incorporated by reference) except thatcompositions of the invention comprising at least two or at least threetriterpenes and excluding cardiac glycoside are evaluated.

Triterpene-based compositions containing oleanolic acid as the primarytriterpene and ursolic acid and betulinic acid as secondary triterpenesmay be found to be active. A composition comprising the threetriterpenes at the following molar ratio(s) may be found to be active:OA:UA:BA is 9-12:0.15-2.5:0.15-2.5. A composition comprising twotriterpenes at the following molar ratio(s) may be found to be active:OA:UA is 9-12:0.33-5 or OA:BA is 9-12:0.33-5.

Example 36 Preparation of Mixtures of Triterpene Forms

The following compositions are made by mixing the specified forms of atriterpene. No specific order of mixing is required. The forms can bemixed in any form in the presence or absence of liquid.

Triterpene Form Present (Y/N) Sample Free acid Salt Derivative ProdrugCombination 1 Y Y N N Combination 2 Y N Y N Combination 3 Y N N YCombination 4 N Y Y N Combination 5 N Y N Y Combination 6 N N Y YCombination 7 N Y N Y Combination 8 Y Y Y N Combination 9 Y N Y YCombination 10 Y Y N Y Combination 11 N Y Y Y Combination 12 Y Y Y  Y.

Each form of triterpene to be included in a combination is provided. Theforms are then mixed to provide the respective combination-basedtriterpene.

For example, triterpene free acid form and triterpene salt form(s) aremixed. Alternatively, triterpene free acid form and triterpenederivative form(s) are mixed. Still, triterpene free acid form andtriterpene prodrug form(s) are mixed.

Example 37 Treatment of Zika Virus Infection in a Subject

Method A. Antiviral Composition Therapy

A subject presenting with Zika virus infection is prescribed antiviralcomposition, and therapeutically relevant doses are administered to thesubject according to a prescribed dosing regimen for a period of time.The subject's level of therapeutic response is determined periodically.The level of therapeutic response can be determined by determining thesubject's Zika virus titre in blood or plasma. If the level oftherapeutic response is too low at one dose, then the dose is escalatedaccording to a predetermined dose escalation schedule until the desiredlevel of therapeutic response in the subject is achieved. Treatment ofthe subject with antiviral composition is continued as needed and thedose or dosing regimen can be adjusted as needed until the patientreaches the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Composition with Another Agent

Method A, above, is followed except that the subject is prescribed andadministered one or more other therapeutic agents for the treatment ofZika virus infection or symptoms thereof. Then one or more othertherapeutic agents can be administered before, after or with theantiviral composition. Dose escalation (or de-escalation) of the one ormore other therapeutic agents can also be done.

Example 38 In Vitro Evaluation of Antiviral Activity Against Zika VirusInfection

Method A. Pure Compound

Vero E6 cells (also known as Vero C1008 cells, ATTC No. CRL-1586;https://www.atcc.org/Products/All/CRL-1586.aspx) were infected with ZIKV(Zika virus strain PRVABC59; ATCC VR-1843;https://www.atcc.org/Products/All/VR-1843.aspx) at an MOI (multiplicityof infection) of 0.2 in the presence of cardiac glycoside. Cells wereincubated with virus and compound for 1 hr, after which the inoculum andcompound were discarded. Cells were given fresh medium and incubated for48 hr, after which they were fixed with formalin and stained for ZIKVinfection. Other compounds are evaluated under the same conditions andexhibit very varying levels of antiviral activity against Zika virus.

Method B. Compound in Extract Form

An extract containing a target compound being tested is evaluated asdetailed in Method A, except that the amount of extract is normalized tothe amount of target compound in the extract. For example, an extractcontaining 2% wt of oleandrin contains 20 microg of oleandrin per 1 mgof extract. Accordingly, if the intended amount of oleandrin forevaluation is 20 microg, then 1 mg of extract would be used in theassay.

Example 39 Treatment of Filovirus Infection in a Subject

Exemplary Filovirus infections include Ebolavirus and Marburgvirus.

Method A. Antiviral Composition Therapy

A subject presenting with Filovirus infection is prescribed antiviralcomposition, and therapeutically relevant doses are administered to thesubject according to a prescribed dosing regimen for a period of time.The subject's level of therapeutic response is determined periodically.The level of therapeutic response can be determined by determining thesubject's Filovirus titre in blood or plasma. If the level oftherapeutic response is too low at one dose, then the dose is escalatedaccording to a predetermined dose escalation schedule until the desiredlevel of therapeutic response in the subject is achieved. Treatment ofthe subject with antiviral composition is continued as needed and thedose or dosing regimen can be adjusted as needed until the patientreaches the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Composition with Another Agent

Method A, above, is followed except that the subject is prescribed andadministered one or more other therapeutic agents for the treatment ofFilovirus infection or symptoms thereof. Then one or more othertherapeutic agents can be administered before, after or with theantiviral composition. Dose escalation (or de-escalation) of the one ormore other therapeutic agents can also be done.

Example 40 Treatment of Flavivirus Infection in a Subject

Exemplary Flavivirus infections include Yellow Fever, Dengue Fever,Japanese Enchephalitis, West Nile Viruses, Zikavirus, Tick-borneEncephalitis, Kyasanur Forest Disease, Alkhurma Disease, Chikungunyavirus, Omsk Hemorrhagic Fever, Powassan virus infection.

Method A. Antiviral Composition Therapy

A subject presenting with Flavivirus infection is prescribed antiviralcomposition, and therapeutically relevant doses are administered to thesubject according to a prescribed dosing regimen for a period of time.The subject's level of therapeutic response is determined periodically.The level of therapeutic response can be determined by determining thesubject's Flavivirus titre in blood or plasma. If the level oftherapeutic response is too low at one dose, then the dose is escalatedaccording to a predetermined dose escalation schedule until the desiredlevel of therapeutic response in the subject is achieved. Treatment ofthe subject with antiviral composition is continued as needed and thedose or dosing regimen can be adjusted as needed until the patientreaches the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Composition with Another Agent

Method A, above, is followed except that the subject is prescribed andadministered one or more other therapeutic agents for the treatment ofFlavivirus infection or symptoms thereof. Then one or more othertherapeutic agents can be administered before, after or with theantiviral composition. Dose escalation (or de-escalation) of the one ormore other therapeutic agents can also be done.

Example 41 Evaluation of Antiviral Activity Against Zikavirus and DengueVirus

A CPE-based antiviral assay was performed by infecting target cells inthe presence or absence of test compositions, at a range ofconcentrations. Infection of target cells by results in cytopathiceffects and cell death. In this type of assay, reduction of CPE in thepresence of test composition, and the corresponding increase in cellviability, is used as an indicator of antiviral activity. For CPE-basedassays, cell viability was determined with a neutral red readout. Viablecells incorporate neutral red in their lysosomes. Uptake of neutral redrelies on the ability of live cells to maintain a lower pH inside theirlysosomes than in the cytoplasm, and this active process requires ATP.Once inside the lysosome, the neutral red dye becomes charged and isretained intracellularly. After a 3-hour incubation with neutral red(0.033%), the extracellular dye was removed, cells were washed with PBS,and the intracellular neutral red was solubilized with a solution of 50%ethanol+1% acetic acid. The amount of neutral red in solution wasquantified by reading the absorbance (optical density) of each well at490 nm

Adherent cell lines were used to evaluate the antiviral activity ofcompositions against a panel of viruses. Compositions were pre-incubatedwith the target cells for 30 min before the addition of virus to thecells. The compositions were present in the cell culture medium for theduration of the infection incubation period. For each infection assay, aviability assay was set up in parallel using the same concentrations ofcompositions (duplicates) to determine cytotoxicity effects of thecompositions in the absence of virus.

The antiviral activity of test compositions was determined by comparinginfection levels (for immunostaining-based assay) or viability (forCPE-based assays) of cells under test conditions to the infection levelor viability of uninfected cells. Cytotoxic effects were evaluated inuninfected cells by comparing viability in the presence of inhibitors tothe viability of mock-treated cells. Cytotoxicity was determined by anXTT viability assay, which was conducted at the same timepoint as thereadout for the corresponding infection assay.

Test compositions were dissolved in 100% methanol. Eight concentrationsof the compositions were generated (in duplicate) by performing 8-folddilutions, starting with 50 M as the highest concentration tested. Thehighest test concentration of composition (50 M) resulted in a 0.25%final concentration of methanol (v/v %) in the culture medium. An 8-folddilution series of methanol vehicle was included in each assay plate,with concentrations mirroring the final concentration of methanol ineach composition test condition. When possible, the EC50 and CC50 of thecomposition was determined for each assay using GraphPad Prism software.

Antiviral activity was evaluated by the degree of protection againstvirus-induced cytopathic effects (CPE). Cells were challenged with virusin the presence of different concentrations of control or compositions.The extent of protection against CPE was monitored after 6 days (ZIKV,Zikavirus) or 7 days (DENV, Dengue virus) post infection by quantifyingcell viability in different test conditions and comparing values withthat of untreated cells and cells treated with vehicle alone (infectionmedium).

Quality controls for the neutralization assay were performed on everyplate to determine: i) signal to background (S/B) values; ii) inhibitionby the known inhibitors, and iii) variation of the assay, as measured bythe coefficient of variation (C.V.) of all data points. Overallvariation in the infection assays ranged from 3.4% to 9.5%, and overallvariation in the viability assays ranged from 1.4% to 3.2%, calculatedas the average of all C.V. values. The signal-to-background (S/B) forthe infection assays ranged from 2.9 to 11.0, while thesignal-to-background (S/B) for the viability assays ranged from 6.5 to29.9.

Protection of DENV2-induced cytopathic effect (CPE) with Neutral Redreadout: For the DENV2 antiviral assay, the 08-10381 Montserrat strainwas used. Viral stocks were generated in C6/36 insect cells. Vero cells(epithelial kidney cells derived from Cercopithecus aethiops) weremaintained in MEM with 5% FBS (MEM5). For both the infection and theviability assays, cells were seeded at 10,000 cells per well in 96-wellclear flat bottom plates and maintained in MEM5 at 37° C. for 24 hours.The day of infection, samples were diluted 8-fold in U-bottom platesusing MEM with 1% bovine serum albumin (BSA). Test material dilutionswere prepared at 1.25× the final concentration and 40 μl were incubatedwith the target cells at 37° C. for 30 minutes. Following the testmaterial pre-incubation, 10 μl of virus dilutions prepared in MEM with1% BSA was added to each well (50 μl final volume per well) and plateswere incubated at 37° C. in a humidified incubator with 5% CO2 for 3hours. The volume of virus used in the assay was previously determinedto produce a signal in the linear range inhibited by Ribavirin andcompound A3, known inhibitors of DENV2. After the infection incubation,cells were washed with PBS, then MEM containing 2% FBS (MEM2) to removeunbound virus. Subsequently, 50 μl of medium containing inhibitordilutions prepared at a 1× concentration in MEM2 was added to each well.The plate was incubated at 37° C. in the incubator (5% CO2) for 7 days.Controls with no virus (“mock-infected’), infected cells incubated withmedium alone, infected cells incubated with vehicle alone (methanol),and wells without cells (to determine background) were included in theassay plate. Control wells containing 50 M Ribavirin and 0.5 M compoundA3 were also included on the assay plate. After 7 days of infection,cells were stained with neutral red to monitor cell viability. Testmaterials were evaluated in duplicates using serial 8-fold dilutions ininfection medium. Controls included cells incubated with no virus(“mock-infected”), infected cells incubated with medium alone, orinfected cells in the presence of Ribavirin (0.5 μM) or A3 (0.5μM). Afull duplicate inhibition curve with methanol vehicle only was includedon the same assay plate.

Protection of ZIKV-induced cytopathic effect (CPE) with Neutral Redreadout: For the ZIKV antiviral assay, the PLCal_ZV strain was used.Vero cells (epithelial kidney cells derived from Cercopithecus aethiops)were maintained in MEM with 5% FBS (MEM5). For both the infection andthe viability assays, cells were seeded at 10,000 cells per well in96-well clear flat bottom plates and maintained in MEM5 at 37° C. for 24hours. The day of infection, samples were diluted 8-fold in U-bottomplates using MEM with 1% bovine serum albumin (BSA). Test materialdilutions were prepared at 1.25× the final concentration and 40 μl wereincubated with the target cells at 37° C. for 30 minutes. Following thetest material pre-incubation, 101 of virus dilutions prepared in MEMwith 1% BSA was added to each well (501 final volume per well) andplates were incubated at 37° C. in a humidified incubator with 5% CO2for 3 hours. After the infection incubation, cells were washed with PBS,then MEM containing 2% FBS (MEM2) to remove unbound virus. Subsequently,501 of medium containing inhibitor dilutions prepared at a 1×concentration in MEM2 was added to each well. The plate was incubated at37° C. in the incubator (5% CO2) for 6 days. Controls with no virus(“mock-infected’), infected cells incubated with medium alone, infectedcells incubated with vehicle alone (methanol), and wells without cells(to determine background) were included in the assay plate. After 6 daysof infection, cells were stained with neutral red to monitor cellviability. Test materials were evaluated in duplicates using serial8-fold dilutions in infection medium. Controls included cells incubatedwith no virus (“mock-infected”), infected cells incubated with mediumalone, or infected cells in the presence of A3 (0.5 M). A full duplicateinhibition curve with methanol vehicle only was included on the sameassay plate.

Analysis of CPE-based viability data: for the neutral red assays, cellviability was determined by monitoring the absorbance at 490 nm. Theaverage signal obtained in wells with no cells was subtracted from allsamples. Then, all data points were calculated as a percentage of theaverage signal observed in the 8 wells of mock (uninfected) cells on thesame assay plate. Infected cells treated with medium alone reduced thesignal to an average of 4.2% (for HRV), 26.9% (for DENV), and 5.1% (forZIKV) of that observed in uninfected cells. The signal-to-background(S/B) for this assay was 2.9 (for DENV), and 7.2 (for ZIKV), determinedas the viability percentage in “mock-infected” cells compared to that ofinfected cells treated with vehicle only.

Viability assay (XTT) to assess compound-induced cytotoxicity:Mock-infected cells were incubated with inhibitor dilutions (or mediumonly) using the same experimental setup and inhibitor concentrations aswas used in the corresponding infection assay. The incubationtemperature and duration of the incubation period mirrored theconditions of the corresponding infection assay. Cell viability wasevaluated with an XTT method. The XTT assay measures mitochondrialactivity and is based on the cleavage of yellow tetrazolium salt (XTT),which forms an orange formazan dye. The reaction only occurs in viablecells with active mitochondria. The formazan dye is directly quantifiedusing a scanning multi-well spectrophotometer. Background levelsobtained from wells with no cells were subtracted from all data-points.Controls with methanol vehicle alone (at 7 concentrations mirroring thefinal percent methanol of each Oleandrin test wells) were included inthe viability assay plate. The extent of viability was monitored bymeasuring absorbance at 490 nm.

Analysis of cytotoxicity data: For the XTT assays, cell viability wasdetermined by monitoring the absorbance at 490 nm. The average signalobtained in wells with no cells was subtracted from all samples. Then,all data points were calculated as a percentage of the average signalobserved in the 8 wells of mock (uninfected) cells on the same assayplate. The signal-to-background (S/B) for this assay was 29.9 (for IVA),8.7 (for HRV), 6.5 (for DENV), and 6.7 (for ZIKV), determined as theviability percentage in “mock-infected” cells compared to the signalobserved for wells without cells.

Example 42 Evaluation of Antiviral Activity Against Filovirus(Ebolavirus and Marburgvirus)

Method A.

Vero E6 cells were infected with EBOV/Kik (A, MOI=1) or MARV/Ci67 (B,MOI=1) in the presence of oleandrin, digoxin or PBI-05204, anoleandrin-containing plant extract. After 1 hr, inoculum and compoundswere removed and fresh medium added to cells. 48 hr later, cells werefixed and immunostained to detect cells infected with EBOV or MARV.Infected cells were enumerated using an Operetta. C) Vero E6 weretreated with compound as above. ATP levels were measured byCellTiter-Glo as a measurement of cell viability.

Method B.

Vero E6 cells were infected with EBOV (A,B) or MARV (C,D). At 2 hrpost-infection (A,C) or 24 hr post-infection (B,D), oleandrin orPBI-05204 was added to cells for 1 hr, then discarded and cells werereturned to culture medium. At 48 hr post-infection, infected cells wereanalyzed.

Method C.

Vero E6 cells were infected with EBOV or MARV in the presence ofoleandrin or PBI-05204 and incubated for 48 hr. Supernatants frominfected cell cultures were passaged onto fresh Vero E6 cells, incubatedfor 1 hr, then discarded (as depicted in A). Cells containing passagedsupernatant were incubated for 48 hr. Cells infected with EBOV (B) orMARV (C) were detected as described previously. Control infection rateswere 66% for EBOV and 67% for MARV.

Example 43 Evaluation of Antiviral Activity Against Togaviridae Virus(Alphavirus: VEEV and WEEV)

Vero E6 cells were infected with Venezuelan equine encephalitis virus(A, MOI=0.01) or Western equine encephalitis virus (B, MOI=0.1) for 18hr in the presence or absence of indicated compounds. Infected cellswere detected as described herein and enumerated on an Operetta.

Example 44 Treatment of Paramyxoviridae Infection in a Subject

Exemplary Paramyxoviridae family viral infections include Henipavirusgenus infection, Nipah virus infection, or Hendra virus infection.

Method A. Antiviral Composition Therapy

A subject presenting with Paramyxoviridae family infection is prescribedantiviral composition, and therapeutically relevant doses areadministered to the subject according to a prescribed dosing regimen fora period of time. The subject's level of therapeutic response isdetermined periodically. The level of therapeutic response can bedetermined by determining the subject's virus titre in blood or plasma.If the level of therapeutic response is too low at one dose, then thedose is escalated according to a predetermined dose escalation scheduleuntil the desired level of therapeutic response in the subject isachieved. Treatment of the subject with antiviral composition iscontinued as needed and the dose or dosing regimen can be adjusted asneeded until the patient reaches the desired clinical endpoint.

Method B. Combination Therapy: Antiviral Composition with Another Agent

Method A, above, is followed except that the subject is prescribed andadministered one or more other therapeutic agents for the treatment ofParamyxoviridae family infection or symptoms thereof. Then one or moreother therapeutic agents can be administered before, after or with theantiviral composition. Dose escalation (or de-escalation) of the one ormore other therapeutic agents can also be done.

As used herein and unless otherwise specified, the term “about” or“approximately” are taken to mean 10%, +5%, ±2.5% or 1% of a specifiedvalued. As used herein and unless otherwise specified, the term“substantially” is taken to mean “to a large degree”, “at least amajority of”, greater than 70%, greater than 85%, greater than 90%,greater than 95%, greater than 98% or greater than 99%.

When a range is specified herein, the range includes all whole andfractional numbers of said range.

The above is a detailed description of particular embodiments of theinvention. It will be appreciated that, although specific embodiments ofthe invention have been described herein for purposes of illustration,various modifications may be made without departing from the spirit andscope of the invention. Accordingly, the invention is not limited exceptas by the appended claims. All of the embodiments disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure.

The invention claimed is:
 1. A method of providing neuroprotection in aneurological condition, disease, or disorder comprising administering toa subject in need thereof a triterpene-based composition comprising acombination of a first triterpene oleanolic acid (OA), a secondtriterpene ursolic acid (UA), and a third triterpene betulinic acid(BA), wherein each triterpene is independently selected at eachoccurrence from the free acid form thereof, salt(s) form thereof,derivative(s) thereof, prodrug(s) thereof and/or combination thereof,and wherein the molar ratio of OA:UA:BA is selected from the groupconsisting of about 10:about 1:about 1, about 9-11:about 0.5-1.5:about0.5-1.5, about 9.5-10.5:about 0.75-1.25:about 0.75-1.25, about9.5-10.5:about 0.8-1.2:about 0.8-1.2, about 9.75-10.5:about0.9-1.1:about 0.9-1.1, about 9-12:about 0.15-2.5:about 0.15-2.5, about9-12:about 0.2-2.5:about 0.2-2.5, about 9-12:about 0.25-2.5:about0.25-2.5, about 9-12:about 0.35-2.5:about 0.35-2.5, about 9-12:about0.45-2.5:about 0.45-2.5, about 9-12:about 0.5-2.5:about 0.5-2.5, about9-12:about 0.16-2:about 0.16-2, about 9-12:about 0.2-2:about 0.2-2,about 9-12:about 0.25-2:about 0.25-2, about 9-12:about 0.25-2:about0.25-2, about 9-12:about 0.45-2:about 0.45-2, about 9-12:about0.5-2:about 0.5-2, about 9-12:about 0.16-1.5:about 0.16-1.5, about9-12:about 0.2-1.5:about 0.2-1.5, about 9-12:about 0.25-1.5:about0.25-1.5, about 9-12:about 0.7-1.5:about 0.35-1.5, about 9-12:about0.45-1.5:about 0.45-1.5, about 9-12:about 0.5-1.5:about 0.5-1.5, about9-12:about 0.16-1:about 0.16-1, about 9-12:about 0.2-1:about 0.2-1,about 9-12:about 0.25-1:about 0.25-1, about 9-12:about 0.35-1:about0.35-1, about 9-12:about 0.45-1:about 0.45-1, about 9-12:about0.5-1:about 0.5-1, about 10:about 0.5-2.5:about 0.5-2.5, about 10:about0.1-1.5:about 0.1-1.5, about 9-12:about 0.25-0.75:about 0.25-0.75, about9.5-10.5:about 0.35-0.7:about 0.35-0.7, about 9.5-10.5:about0.4-0.6:about 0.4-0.6, and about 9.75-10.5:about 0.45-0.6:about0.45-0.6; wherein said neurological condition, disease or disorder isselected from the group consisting of Alzheimer's disease, Parkinson'sdisease, stroke, Huntington disease, a taupathy, and a condition havingan etiology associated with excessive proteolysis of amyloid betaprecursor protein, with accumulation of amyloid beta protein in thesynapses of the neurons of a subject, with formation of amyloid fibrilsin the synapses of the neurons of a subject, or with formation ofamyloid plaques in the synapses of the neurons of a subject, amyotrophiclateral sclerosis, bovine spongiform encephalopathy, multiple sclerosis,diabetic neuropathy, autism, juvenile neuronal ceroid lipofuscinosis,stroke, a tauopathy, a neurodegenerative disease having an etiologyassociated with an imbalance in the Tau3R/Tau4R ratio in a subject,Down's syndrome, Pick's disease, corticobasal degeneration, prionsdisease, progressive supranuclear palsy, and frontotemporal dementia. 2.The method of claim 1, wherein said first triterpene is present in atleast four-fold molar excess over said second triterpene or said thirdtriterpene.
 3. The method of claim 1, wherein at least one of saidtriterpenes is present as a mixture of two or more different formsthereof.
 4. The method of claim 3, wherein at least one of saidtriterpenes is present as a mixture of two or more forms defined asfollows: Triterpene Form Present (Y/N) Sample Free acid Salt DerivativeProdrug Combination 1 Y Y N N Combination 2 Y N Y N Combination 3 Y N NY Combination 4 N Y Y N Combination 5 N Y N Y Combination 6 N N Y YCombination 7 N Y N Y Combination 8 Y Y Y N Combination 9 Y N Y YCombination 10 Y Y N Y Combination 11 N Y Y Y Combination 12 Y Y Y  Y.


5. The method of claim 1, wherein a) oleanolic acid is present in molarexcess over the combined total moles of ursolic acid and betulinic acid,and ursolic acid and betulinic acid are present at about the same molarcontent; b) oleanolic acid is present in molar excess over the combinedtotal moles of ursolic acid and betulinic acid, and ursolic acid can bepresent in molar excess over betulinic acid; c) oleanolic acid ispresent in molar excess over the combined total moles of ursolic acidand betulinic acid, and betulinic acid can be present in molar excessover ursolic acid; d) oleanolic acid is present in molar excess overursolic acid, and ursolic acid can be present in molar excess overbetulinic acid; or e) oleanolic acid is present in molar excess overbetulinic acid, and betulinic acid can be present in molar excess overursolic acid.
 6. The method of claim 1, wherein the molar content ofursolic acid approximates that of betulinic acid.
 7. The method of claim1, wherein the molar ratio of OA:UA BA is about 9-12:about 0.2-2.5:about0.2-2.5.
 8. The method of claim 1, wherein the composition comprises asthe primary pharmacologically active components oleanolic acid and/orsalt thereof, ursolic acid and/or salt thereof, and betulinic acidand/or salt thereof, wherein the molar ratio of OA:UA:BA is about9-12:about 0.2-2.5:about 0.2-2.5.
 9. The method of claim 1, wherein saidcomposition excludes a cardiac glycoside, steroid, and pharmacologicallyactive polysaccharide.
 10. The method of claim 1, wherein saidcomposition further comprises one or more other therapeuticallyeffective agents.
 11. The method of claim 9, wherein the one or moreother therapeutically effective agents is selected from the groupconsisting of BACE inhibitor (beta-secretase 1, beta-site amyloidprecursor protein cleaving enzyme 1, beta-site APP cleaving enzyme 1,membrane-associated aspartic protease 2, memapsin-2, aspartyl protease2, and ASP2), AZD3293, acetylcholinesterase inhibitors, Namenda™(memantine HCl), Aricept™ (donepezil), Razadyne™ (galantamine), Exelon™(rivastigmine), Cognex™ (tacrine), anticonvulsants, NMDA (n-methyld-aspartate) receptor antagonists, sodium channel blockers Vitamin E,Baclofen (a derivative of CoQ10), Lamotrigine (an anticonvulsant),remacemide (an anesthetic which is a low affinity NMDA antagonist),riluzole (Na channel blocker), Alteplase (a thrombolytic agent),levodopa, carbidopa, amantadine, COMT (catechol O-methyl transferase)inhibitor, tolcapone, entacapone, opicapone, dopamine agonist,bromocriptine, pergolide, pramipexole, ropinirole, piribedil,cabergoline, apomorphine, lisuride, MAO-B (monoamine oxidase-B)inhibitor (selective and non-selective MAO-B inhibitors),anticholinergic, cholinesterase inhibitor, isocarboxazid, nialamide,phenelzine, hydracarbazine, rasagiline, selegiline, linezolid, or acombination thereof.
 12. The method of claim 1, wherein said compositioncomprises as the primary pharmacologically active components oleanolicacid and/or salt thereof (OA); ursolic acid and/or salt thereof (UA);and betulinic acid and/or salt thereof (BA), wherein the molar ratio ofOA:UA:BA is about 9-12:about 0.2-2.5:about 0.2-2.5.
 13. The method ofclaim 12, wherein said composition further comprises at least onepharmaceutical excipient.
 14. The method of claim 1, wherein saidadministering is conducted on a recurring basis over an extended period,wherein: a) the recurring basis is daily, every other day, every secondday, every third day, every fourth day, every fifth day, every sixthday, weekly, every other week, every second week, every third week,monthly, bimonthly, semi-monthly, every other month every second month,quarterly, every other quarter, trimesterly, seasonally, semi-annuallyand/or annually; b) the extended period is one or more weeks, one ormore months, one or more quarters and/or one or more years; and/or c)the effective dose is administered one or more times in a day.